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PubMed1986

Assessing the delivery of neutrophils to tissues in neutropenia.

Studies of neutrophil kinetics in neutropenic individuals, as well as clinical observations of variability in the occurrence of infection among patients with neutropenia, have suggested that blood neutrophil counts may not uniformly reflect the effective delivery of neutrophils to extravascular tissues where the cells perform their principal host defense functions. To evaluate this possibility we developed a sensitive, reproducible method of measuring the extravascular delivery of neutrophils to a normal mucosal site of neutrophil turnover. This method is based upon the quantification of neutrophils recoverable from saline mouth wash specimens. Twenty-five mL specimens, obtained in a controlled manner from neutropenic patients and normal subjects, were centrifuged and the sediments resuspended in 1.0 mL Hank's buffer with 2 micrograms acridine orange, incubated at 37 degrees C for 15 minutes, and then examined in a hemocytometer chamber by fluorescence microscopy. Neutrophils could be clearly distinguished by their characteristic fluorescence and were counted. With this method as few as 1,500 neutrophils were detected reliably in mouth wash specimens. Mucosal neutrophil counts varied less than 10% with repeated sampling of individual subjects over 5-day periods and were consistently greater than 1.3 X 10(5)/specimen in non-neutropenic individuals. Although profound neutropenia was generally reflected by lower than normal oral mucosal neutrophil counts, these counts were significantly higher in individuals with chronic severe neutropenia (blood neutrophils less than 300/mm3) than in patients with acute neutropenia of comparable severity that had developed following chemotherapy. Also, in individuals recovering from profound neutropenia, neutrophils usually reappeared earlier in mouth wash specimens than in blood, and oral mucosal neutrophil counts attained recovery levels more rapidly than did blood counts. This phenomenon was particularly evident in an individual with cyclic neutropenia. Moreover, mucosal neutrophils could occasionally be detected in profoundly neutropenic patients when neutrophils were not present in blood samples. These findings indicate that mucosal neutrophil counts in individuals with neutropenia provide information about the delivery of neutrophils to tissues that may not be apparent from blood neutrophil counts alone.

Acute Disease↗
PubMed1998

Platelet-neutrophil interactions during hemodialysis: a proposed biocompatibility approach.

Platelet interaction with neutrophils may occur to a significant degree during hemodialysis (HD). We have recently shown that the enhanced neutrophil reactive oxygen species (ROS) production during the early phase of HD with cuprophan (CUP) is sustained by neutrophils which have bound platelets through P-selectin (CD62P). The evaluation of platelet-neutrophil interactions during dialysis offers the novel aspect of cell-cell interactions as a new parameter for studying the biocompatibility of dialyzer membranes. By the use of flow cytometry techniques, the present study was set up to analyze intradialytic platelet-neutrophil coaggregate formation and neutrophil ROS (hydrogen peroxide) production from 6 HD patients each dialysed with CUP, cellulose diacetate (CDA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN) in a cross-over clinical trial. Platelet-neutrophil coaggregate formation (percentage of neutrophil cells positive for CD62P) and ROS production by neutrophils (total population; CD62P+ cells; CD62P- cells) were determined before HD and after 10', 20'and 40'. CD62P+ neutrophils significantly increased during HD with CUP (10', 20', 40'), PMMA (20') and CDA (20), while no change was observed with PAN. The difference between CUP and the other membranes was significant at 10', 20' and 40'; at 20', PMMA vs PAN p<0.005. ROS production by total neutrophil population significantly increased with CUP (10', 20), PMMA (20) and CDA (20'). The increase with CUP was higher at 10' when compared to CDA (p<0.020) or PAN (p<0.005), and at 20' versus the other three membranes; at 20' PMMA vs PAN p<0.005. Only neutrophils gated in neutrophil-platelet coaggregate areas (CD26P+ neutrophils) produced hydrogen peroxide. ROS production by CD62P+ neutrophils significantly increased with CUP (10', 20), PMMA (20') and CDA (20'). The increase with CUP was significantly (p<0.0002) higher than the other three membranes at 10' and 20'; at 20', PMMA vs PAN p<0.02. With each membrane, ROS production by CD62P- neutrophils showed no significant change at any time point during HD. The results of the present study indicate that interactions between platelets and neutrophils can mediate some pathophysiological abnormalities associated with hemodialysis treatment. Our data show that cellulose diacetate, a modified cellulosic membrane, exhibits a biocompatibility profile in terms of platelet-neutrophil interactions improved as compared to the parent cellulose membrane and comparable to that of some synthetic membranes. Our data also show that there is considerable variability in the biocompatibility of synthetic membranes. Though cellulosic membranes are generally considered as being less biocompatible than synthetic membranes, our results indicate that classification of membranes by biocompatibility is more complex than a simple division into cellulosic and synthetic membranes, especially with the advent of modified cellulosic membranes.

Acrylic Resins↗
PubMed1998

P-Selectin support of neonatal neutrophil adherence under flow: contribution of L-selectin, LFA-1, and ligand(s) for P-selectin.

To further define the neonatal neutrophil's ability to localize to inflamed tissue compared with adult cells, we examined the neonatal neutrophil interactions with P-selectin monolayers under two conditions: (1) attachment under constant shear stress and flow and (2) detachment where cells were allowed to attach in the absence of shear stress and then shear stress is introduced and increased in step-wise increments. Cord blood and adult neutrophils had minimal interactions with unstimulated human umbilical vein endothelial cells (HUVECs) at a constant shear stress of 2 dynes/cm2. There was a marked increase in the number of both neonatal and adult cells interacting (interacting cells = rolling + arresting) with HUVECs after histamine stimulation, although the neonatal value was only 40% of adult (P < .05). Neonatal neutrophils also had significantly decreased interaction with monolayers of Chinese hamster ovary (CHO) cells transfected with human P-selectin (CHO-P-selectin; 60% of adult values, P < .003). Of the interacting cells, there was a lower fraction of neonatal cells that rolled compared with adult cells on both stimulated HUVECs and CHO-P-selectin. That neonatal neutrophil L-selectin contributes to the diminished attachment to P-selectin is supported by the following: (1) Neonatal neutrophils had significantly diminished expression of L-selectin. (2) Anti-L-selectin monoclonal antibody reduced the number of interacting adult neutrophils to the level seen with untreated neonatal neutrophils, but had no effect on neonatal neutrophils. In contrast, L-selectin appeared to play no role in maintaining the interaction of either neonatal or adult neutrophils in the detachment assay. Once attachment occurred, the neonatal neutrophil's interaction with the P-selectin monolayer was dependent on LFA-1 and to other ligands to a lesser degree based on the following: (1) Control neonatal neutrophils had decreased rolling fraction compared with adult neutrophils, although the total number of interacting neutrophils was equal between groups. (2) Anti-LFA-1 treatment resulted in an increase in the rolling fraction of both neonatal and adult neutrophils. However, whereas the number of interacting adult neutrophils remained unchanged, the number of neonatal neutrophils decreased with increased shear stress. We speculate that this increased detachment of neonatal cells is due to differences in neutrophil ligand(s) for P-selectin.

Adult↗
PubMed1996

Effect of Thoracotomy and Cardiopulmonary Bypass on Activated Platelet and Neutrophil Dynamics and Platelet Emboli in a Pig Model.

The effects of thoracotomy and components of extracorporeal circuits on dynamics of platelets and neutrophils were quantified with autologous In-111-labeled platelets (INPLT) and neutrophils (INN) during cardiopulmonary bypass (CPB) operations in Yorkshire pigs. Cardiopulmonary bypass was carried out with a hollow-fiber oxygenator and an arterial filter in 48 pigs (30-35 kg; 12 unoperated controls for platelets and neutrophils; 12 sham operated controls; 12 with 180 minutes of CPB with platelets and neutrophils; 12 with 90 minutes of CPB and 90 minutes of reperfusion at 2.5-3.5 one/min. Platelets and neutrophils were labeled with In-111 tropolone and were injected intravenously: platelets at 24 hours and neutrophils at 15 minutes before CPB. All pigs were systemically heparinized [activated coagulation time (ACT) > 400 seconds]; CPB was instituted with a roller pump, oxygenator (OX; Bentley Univox, 1.8 m2), and arterial filter (AF; 0.025 m2) for durations of 180 minutes and 90 minutes of bypass, followed by 90 minutes of reperfusion. The kinetics and pooling of platelets and neutrophils were monitored by a Geiger probe. The adherent thrombi and neutrophils in the OX, AF, viscera, and brain were imaged with a gamma camera and were measured with an ion chamber and a gamma counter. The percentile distribution of labeled platelets and neutrophils expressed as the mean +/- standard deviation of injected dose in eight groups was calculated and statistical analyses were performed (ANOVA and paired t-test). Sham operation alone increased platelet retention in the lung, heart, and brain significantly (p < 0.001) over that of unoperated pigs. Neutrophil margination to lung immediately after injection was high; CPB and reperfusion altered the distribution in blood, viscera, and connective tissues. During CPB, an equilibrium among single platelets, platelet thrombi, and emboli was reached in the blood, oxygenator, arterial filter, perfused organs, and tissues. After CPB, the pulmonary neutrophil retention increased significantly (p < 0.001). Reperfusion of 90 minutes following 90 minutes of CPB decreased the level of neutrophils and increased the level of platelets in the lung. Only a small amount of platelets and neutrophils was retained in the oxygenator and arterial filter. Neutrophil retention in the OX and AF was higher than that of platelets. The small amount of retained neutrophils in the heart, kidneys, and brain suggested that cytokines, rather than marginated neutrophils alone, may play a major role in inflammatory insult to these organs during and after CPB. OX thrombi increased with the time of CPB; AF thrombus in both groups was almost similar. During CPB, AF functioned minimally as a thrombus trap with a small percent of retained thrombi; reperfusion post-CPB did not change the amount. Thoracotomy alone has a significant effect on platelet and neutrophil kinetics, and on the subsequent effect of thrombus formation, embolization, and neutrophil margination in organs during the CPB procedure.

Journal Article↗
PubMed1992

Induction of neutrophil infiltration by rat chemotactic cytokine (CINC) and its inhibition by dexamethasone in rats.

In vivo effects of cytokine-induced neutrophil chemotactic factor (CINC) derived from rats on neutrophil infiltration were investigated using an air-pouch-type inflammation model in rats, and effects of dexamethasone on neutrophil infiltration induced by CINC was also examined in order to gain further insight into the mechanism of antiinflammatory activity of glucocorticoids. Injection of CINC into the air pouch made on the dorsum of rats induced a marked infiltration of neutrophils into the pouch fluid but not mononuclear cells and eosinophils during a 30-min interval after the injection. Maximum effect was induced at a dose of 1.4 micrograms/pouch. Treatment with dexamethasone 3 h before the injection of CINC suppressed the neutrophil infiltration in a dose-dependent manner, but no complete inhibition was observed. CINC injection into the air pouch of rats that had been sacrificed by bleeding in order to minimize neutrophil infiltration from blood stream also stimulated neutrophil infiltration into the pouch fluid when the carcass was incubated at 37 degrees C for 30 min, but the number of infiltrated neutrophils was about 35% of CINC-induced neutrophil infiltration in intact rats. CINC-induced neutrophil infiltration in the carcass, which is supposed to be a reflection of neutrophil migration from extravascular space in subcutaneous tissues to pouch fluid, was not inhibited by dexamethasone treatment. Therefore, the inhibition of neutrophil infiltration by dexamethasone might be due to inhibition of the extravasation of peripheral neutrophils but not due to inhibition of neutrophil chemotaxis from subcutaneous extravascular space to pouch fluid. These findings suggest that clinical effects of steroidal antiinflammatory drugs on neutrophil infiltration in inflammatory disease is partly due to inhibition of neutrophil extravasation induced by preformed neutrophil chemotactic factors in the inflammatory site.

Animals↗
PubMed1997

The role of neutrophil elastase in human pulmonary artery endothelial cell injury.

Neutrophils are thought to play a key role in tissue injury. We investigated the role of human neutrophil-derived elastase in the induction of injury to human pulmonary artery endothelial cells. Incubation of endothelial cells with neutrophils increased the release of lactate dehydrogenase activity, thrombomodulin, and preloaded fura-2 from endothelial cells, indicating that neutrophils induce endothelial cell injury. Attachment alone of neutrophils to endothelial cells appeared to induce activation because elastase release and N-formyl-mentionyl-leucyl-phenylalanine (fMLP)-induced superoxide (O2) production from neutrophils incubated with endothelial cells were greater than from neutrophils only. When endothelial cell were incubated with neutrophils stimulated by fMLP or phorbol myristate acetate, the amount of elastase in the medium and endothelial cell damage was further enhanced. However, when neutrophils were blocked from direct attachment to endothelial cells using a membrane filter, endothelial cell damage was ameliorated, while exogenous neutrophil elastase and medium containing neutrophil-released elastase did not induce endothelial cell injury. An inhibitor of neutrophil elastase, ONO-5046 Na, as well as erythromycin, which reduces neutrophil-derived elastase, dramatically inhibited neutrophil-induced endothelial cell injury. Superoxide dismutase (SOD) partially inhibited injury. Injury was completely inhibited by treatment with a combination of ONO-5046 Na and SOD. These results suggest that attachment of neutrophils to endothelial cells is important for endothelial cell damage and that neutrophil-derived elastase plays an important role in endothelial cell injury in combination with O2. In addition, ONO-5046 Na and erythromycin may be useful in treating diseases worsened by excessive neutrophil activity.

Endothelium, Vascular↗
PubMed1989

111Indium-labeled neutrophil migration into the lungs of bleomycin-treated rabbits assessed noninvasively by external scintigraphy.

Factors controlling neutrophil migration into the lung are poorly understood, but their identification is important for our understanding of the pathogenesis of inflammatory lung diseases. Pulmonary inflammation is difficult to quantify, and neutrophils in tissues and BAL may not accurately represent cell migration. In this study, intravenously delivered pulses of rabbit neutrophils labeled with Indium-111 (111In-neutrophils) were used to monitor neutrophil migration into the lungs. Radioactivity quantified in the lung "region of interest" (ROI) of external gamma camera scintigrams recorded 24 h after intravenous 111In-neutrophil injection accurately reflected the actual neutrophil-associated lung tissue radioactivity. ROI radioactivity at 24 h also correlated closely with the percent of 111In-neutrophils that had migrated into lavageable air spaces, and this parameter therefore provided an index of total lung 111In-neutrophil migration. Using 24-h ROI radioactivity and percent of injected 111In-neutrophils recovered in BAL at 24 h as indices of neutrophil migration into the lung, it was found that intratracheal saline caused only a transient neutrophil migration, whereas 10 U/kg intratracheal bleomycin induced migration that persisted for as long as 3 wk. 111In-neutrophil migration into the lung, assessed by external scintigraphy, correlated with total neutrophils quantified in histologic sections (r = 0.71, p = 0.006). The data suggest that this approach will be valuable in investigating mechanisms controlling neutrophil migration in lung inflammation, and that 111In-neutrophil scintigraphy may provide a noninvasive index of total lung neutrophil load that might be useful in staging inflammation in patchy diseases such as idiopathic pulmonary fibrosis.

Animals↗
PubMed2003

Science review: Cell membrane expression (connectivity) regulates neutrophil delivery, function and clearance.

As the principal cellular component of the inflammatory host defense and contributor to host injury after severe physiologic insult, the neutrophil is inherently coupled to patient outcome in both health and disease. Extensive research has focused on the mechanisms that regulate neutrophil delivery, function, and clearance from the inflammatory microenvironment. The neutrophil cell membrane mediates the interaction of the neutrophil with the extracellular environment; it expresses a complex array of adhesion molecules and receptors for various ligands, including mediators, cytokines, immunoglobulins, and membrane molecules on other cells. This article presents a review and analysis of the evidence that the neutrophil membrane plays a central role in regulating neutrophil delivery (production, rolling, adhesion, diapedesis, and chemotaxis), function (priming and activation, microbicidal activity, and neutrophil-mediated host injury), and clearance (apoptosis and necrosis). In addition, we review how change in neutrophil membrane expression is synonymous with change in neutrophil function in vivo. Employing a complementary analysis of the neutrophil as a complex system, neutrophil membrane expression may be regarded as a measure of neutrophil connectivity, with altered patterns of connectivity representing functionally distinct neutrophil states. Thus, not only does the neutrophil membrane mediate the processes that characterize the neutrophil lifecycle, but characterization of neutrophil membrane expression represents a technology with which to evaluate neutrophil function.

Antigens, CD↗
PubMed2000

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils.

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.

Actins↗
PubMed1999

Ethanol-induced neutrophil apoptosis is mediated through nitric oxide.

Clinical reports indicate that acute ethanol intoxication in chronic ethanol abusers is associated with neutropenia. We hypothesize that ethanol accelerates the apoptosis of neutrophils thus decreasing the peripheral blood count of neutrophils. We studied the effect of ethanol on neutrophil apoptosis in vivo as well as in vitro. Human neutrophils harvested from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 0.5+/-0.25 vs. after, 26.1+/-2.6% apoptotic neutrophils/field). Peritoneal neutrophils isolated from ethanol-treated rats also showed increased (P < 0.0001) apoptosis when compared with neutrophils isolated from control rats (control, 0.8+/-0.2% vs. ethanol, 11.8+/-0.7% apoptotic neutrophils/field). In in vitro studies, ethanol in concentrations of 50 mM and higher accelerated the apoptosis of human and rat neutrophils. This effect of ethanol on human neutrophils was time dependent. DNA isolated from ethanol-treated human neutrophils displayed integer multiples of 180 base pairs (ladder pattern), further confirming the effect of ethanol on neutrophil apoptosis. N(G)-monomethyl-L-arginine monoacetate and N(G)-nitro-L-arginine methyl ester, inhibitors of nitric oxide (NO) synthase, attenuated the ethanol-induced neutrophil apoptosis. Sodium nitroprusside, a NO donor, also promoted neutrophil apoptosis. Moreover, ethanol enhanced neutrophil expression of inducible NO synthase. In addition, ethanol stimulated neutrophil NO generation. These results suggest that ethanol accelerates neutrophil apoptosis. This effect of ethanol on neutrophil apoptosis seems to be mediated through the generation of NO.

Animals↗
PubMed1999

Concomitant increase in neutrophil adhesion to inflammatory peritoneum and remote organs during peritonitis.

BACKGROUND: Neutrophils contribute to the host defense mechanism, but they can cause remote organ injury in peritonitis. The purpose of this study was to examine neutrophil adhesion to the peritoneum and remote organs simultaneously in peritonitis using a fluorescence microscopic method. STUDY DESIGN: Experiment 1: Sprague-Dawley rats (n = 16) were injected intraperitoneally (ip) with saline solution or 10(5), 10(7), or 10(9) Escherichia coli. Five hours after challenge, 1 x 10(6) fluorescein-labeled neutrophils were infused. Two minutes after neutrophil injection, five peritoneal samples (the greater omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. Lung myeloperoxidase (MPO) activity was also determined. Experiment 2: Rats (n = 23) were given 10(9) E. coli ip. Before challenge (0 h) or at 1, 5, or 10 h after challenge, labeled neutrophils were infused. Then, the labeled neutrophil numbers in organs and lung MPO activities were assessed as described for Experiment 1. Hemodynamic and arterial blood gas data were also obtained in another set of rats before and at 1, 5, 8 and 10 h after 10(9) E. coli ip challenge. RESULTS: Experiment 1: The labeled neutrophil numbers in the peritoneum, lungs, and kidney showed significant positive correlations with the injected bacterial numbers. Lung MPO also positively correlated with E. coli number and labeled neutrophil number in the lungs. Experiment 2: Labeled neutrophil numbers in the peritoneum and kidney peaked at 5 h. The pulmonary labeled neutrophil number rose, reaching a plateau at 5 h. No remarkable change was observed in the hepatic labeled neutrophil number. There was a positive correlation between lung MPO activity and pulmonary labeled neutrophil number. Hemodynamic and blood gas data reflected a hyperdynamic state. CONCLUSIONS: Concomitant dose-dependent increases in neutrophil adhesion in the peritoneum, lungs, and kidney were observed in this peritonitis model. Increased neutrophil adhesion was transient in the peritoneum and kidney but persistent in the lungs. Strategies modulating neutrophil adhesion in organs are anticipated to be useful for the treatment of peritonitis.

Animals↗
PubMed2000

Increased expression of matrix metalloproteinase-9 in neutrophils in glycogen-induced peritoneal inflammation of guinea pigs.

OBJECTIVE: Matrix metalloproteinase (MMP)-9 plays an important role in neutrophil extravasation and migration by its ability to degrade the major components of basement membrane. To evaluate the expression of neutrophil MMP-9 under inflammatory conditions, we examined the levels of MMP-9 and its mRNA in neutrophils of glycogen-induced peritoneal inflammation. MATERIALS AND METHODS: Male Hartley guinea pigs weighing 250-300 g were intraperitoneally injected with 0.17% glycogen solution, and 13-15 h after the injection, blood and peritoneal neutrophils were isolated. The levels of MMP-9 and its mRNA were analyzed by gelatin zymography and Northern blotting, respectively. Furthermore, MMP-9 activities in the peritoneal supernatants were measured. RESULTS: MMP-9 level in peritoneal neutrophils was essentially the same as that in blood neutrophils, although peritoneal neutrophils were assumed to have extracellularly released MMP-9 from the granules during infiltration into the peritoneal cavity. Interestingly, MMP-9 mRNA was expressed more abundantly in peritoneal neutrophils than in blood neutrophils (p<0.01). Moreover, MMP-9 levels in blood and peritoneal neutrophils were reduced to 30-45% of non-treated controls by actinomycin D (500 microg/kg) or cycloheximide (10 mg/kg)-treatment (p<0.05). In contrast, MMP-9 activity increased in the peritoneal supernatants of glycogen-injected animals was not significantly affected by actinomycin D- or cycloheximide-treatment. In addition, when blood neutrophils of non-injected animals were stimulated with 10(-7) M N-formyl-Met-Leu-Phe, 10 microg/ml lipopolysaccharide, 10 ng/ml phorbol 12-myristate 13-acetate, 10(-8) M IL-8 or 100 U/ml tumor necrosis factor-a, expression of MMP-9 mRNA was markedly increased (p<0.05). CONCLUSIONS: The present observations indicate that MMP-9 gene is transcribed, and MMP-9 protein is synthesized in neutrophils during glycogen-injected peritoneal inflammation. Moreover, it is likely that MMP-9 protein in the peritoneal supernatants is mostly derived from preformed MMP-9 which is stored in the neutrophil granules and extracellularly released during infiltration of neutrophils into the peritoneal cavity. Finally, the transcription of MMP-9 gene can be upregulated in neutrophils by stimulation with inflammatory mediators, even after neutrophils have been matured.

Animals↗
PubMed1988

Impact of nicotine on myocardial neutrophil uptake.

Excessive cigarette smoking is recognized as a major risk factor for ischemic heart disease. Although the mechanism by which smoking enhances this risk is not known, multiple lines of indirect evidence suggest that an adverse effect of nicotine on the interaction between neutrophils and the myocardium may play a central pathogenic role. Accordingly, this study employed an isolated rabbit heart preparation perfused at a constant flow rate with physiologic salt solution containing autologous neutrophils to test the hypotheses that nicotine promotes myocardial neutrophil uptake and that an augmented myocardial neutrophil burden intensifies the actions of stimulated neutrophils on the coronary circulation. Addition of 10(-7) M nicotine to the perfusion medium caused an abrupt and sustained sequestration of 111In-labeled neutrophils by isolated rabbit hearts. In contrast, preincubation of neutrophils in 10(-7) M nicotine without inclusion of the alkaloid in the perfusion medium failed to promote neutrophil uptake. Nicotine neither enhanced myocardial neutrophil sequestration induced by perfusion with hypoxic medium nor potentiated neutrophil chemotactic responses evoked by leukotriene B4, the putative mediator of hypoxia-induced myocardial neutrophil uptake. In addition, nicotine failed to influence either baseline levels or the hypoxia-induced accumulation of immunoreactive leukotriene B4 detected in myocardial biopsies. The inflammatory cell stimulant, formylmethionyl-leucyl-phenylalanine (fMLP), increased coronary vascular resistance in neutrophil-perfused hearts but not in hearts perfused with neutrophil-free medium. The magnitude of the fMLP-induced coronary response was augmented when the myocardial neutrophil burden was increased by the addition of nicotine to the perfusion medium or by perfusion with hypoxic medium. These observations suggest that nicotine promotes myocardial neutrophil uptake by mechanisms that do not relate to enhanced release and/or effects of endogenous leukotriene B4 and that sequestration of neutrophils intensifies their actions on the coronary circulation.

Animals↗
PubMed1999

Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation.

Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the major cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by measuring elastinolytic activity whereas human neutrophil elastase content has been determined using immunological techniques. Neutrophils contain several intracellular protease inhibitors therefore measurement of elastase activity may underestimate the total NE content. The aim of the current study was to develop immunological techniques to allow investigation of the cellular content, distribution and release of ENE from purified equine neutrophils. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in equine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main inhibitor of elastase were found to be present at 0.813 pg +/- 0.179 and 0.021 pg +/- 0.003 (mean +/- SEM, n = 11 individual horses) per neutrophil, respectively. This represents twice as much elastase as previously found in the equine neutrophil and a comparable amount to that reported in human neutrophils. Immunolocalisation demonstrated that ENE 2A has a granular distribution within the cytosol of neutrophils, whereas API exhibits a uniform non-granular cytoplasmic appearance. In addition the kinetics of simultaneous generation and release of superoxide anions (SOA) and release of ENE 2A from equine neutrophils, stimulated in vitro by zymosan-activated serum (ZAS) in the presence and absence of the cation chelator ethylene glycol-N,N,N',N'-tetraacetic acid (EGTA), showed a close relationship between total SOA generation and total ENE 2A release during the initial 90 min post-ZAS stimulation and the dependence of both events on extracellular cations. In conclusion these studies have shown that horse and human neutrophil elastase content and mediator release functions are more closely matched than was previously thought. This suggests that the species differences in pathology resulting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and extra-cellular protease inhibitors.

Animals↗
PubMed1996

Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation.

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.

Animals↗
PubMed1996

Contact-induced modulation of neutrophil elastase secretion and phagocytic activity by platelets.

It has been reported that platelets stimulate generation of reactive oxygen species in neutrophils and monocytes by a mechanism that requires mutual cell-cell contact and the presence of P-selectin on the platelet surface. In the present study we investigated the effect of platelet-neutrophil contacts on neutrophil elastase secretion and phagocytic activity. Non-activated or thrombin-activated platelets were fixed with formaldehyde, washed and incubated with neutrophils in the absence or presence of various neutrophil agonists. Elastase secretion was determined by measuring the enzyme activity in cell-free supernatants using a chromogenic substrate. Platelet-neutrophil adhesion and ingestion of zymosan particles by neutrophils were quantitated by light microscopy. Platelets significantly reduced elastase secretion from neutrophils but had no effect on the elastase activity in the supernatant of neutrophil lysates. When neutrophils were stimulated with the ionophore A23187 or the chemotactic peptide FMLP, thrombin-activated platelets were more potent to inhibit elastase secretion when compared with non-activated platelets. Neutrophils that were not able to bind platelets to their surface had a significantly lower phagocytic activity when compared with neutrophil with adherent platelets or neutrophils that were incubated in the absence of platelets. The results indicate that platelet-neutrophil contacts may also lead to an inhibition of neutrophil functions and that such inhibition could be due to a transient contact rather than due to a firm platelet-neutrophil adhesion.

Blood Platelets↗
PubMed1998

Effects of proinflammatory cytokines and bacterial toxins on neutrophil rheologic properties.

OBJECTIVE: To examine the changes in neutrophil deformability, aggregation, and adherence in response to stimulation with proinflammatory cytokines and bacterial toxins. DESIGN: Prospective, randomized trial. SETTING: Research laboratory. SUBJECTS: Neutrophils isolated from healthy volunteers. INTERVENTIONS: Neutrophils were exposed to tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-8, their combination, endotoxin (LPS), lipoteichoic acid (LTA), and staphyloccocal enterotoxin B (SEB). Neutrophil deformability was measured as percent neutrophils filtered through 5-microm diameter filters. Aggregation was measured using a platelet aggregometer. Adherence was determined by examining the binding of neutrophils to albumin-coated latex beads. MEASUREMENTS AND MAIN RESULTS: Exposure to TNF-alpha and IL-1beta led to significant decreases in neutrophil filterability, which was attenuated by cytochalasin D pretreatment. LPS and LTA also decreased deformability, suggesting that these toxins directly stimulated neutrophils independent of cytokines. IL-8 and SEB did not significantly affect neutrophil deformability. TNF-alpha and LPS were associated with significant neutrophil aggregation, which was inhibited by pretreatment with anti-CD18 antibodies. Neutrophil aggregation was not affected by IL-1beta, LTA, or SEB. TNF-alpha, IL-8, and LPS increased neutrophil adherence, which also was attenuated by pretreatment with anti-CD18 antibodies. IL-1beta, LTA, and SEB did not significantly affect neutrophil adherence. CONCLUSIONS: Cytokines and bacterial toxins differ in their effects on neutrophil deformability, aggregation, and adherence. Of the cytokines examined, TNF-alpha appears to have the greatest direct effects on neutrophil rheology. Similarly, endotoxin appears to have greater direct effects on neutrophil rheology than the Gram-positive bacterial toxins, LTA, and staphylococcal enterotoxins.

Cell Adhesion↗
PubMed2000

CD44-mediated neutrophil apoptosis in the rat.

BACKGROUND: Apoptosis is an important mechanism by which neutrophils are removed from sites of inflammation, including the kidney. This study investigated whether ligation of the cell-surface adhesion molecule, CD44, can trigger neutrophil apoptosis. METHODS: The anti-rat CD44 antibody OX-50 was used to induce apoptosis of cultured blood neutrophils, as determined by flow cytometry using annexin V staining and by transmission electron microscopy. The functional consequences of OX-50-mediated neutrophil depletion were examined in a rat model of accelerated antiglomerular basement membrane glomerulonephritis. RESULTS: Flow cytometric analysis using the OX-50 antibody, which recognizes the common amino terminal domain of CD44, showed that rat blood neutrophils express very high levels of CD44. The addition of OX-50, but not control antibodies, rapidly induced neutrophil apoptosis in cultured rat blood leukocytes, as demonstrated by annexin V staining and by electron microscopy. Cross-linking of CD44 was essential since F(ab) fragments of the OX-50 antibody failed to induce neutrophil apoptosis. The CD44 ligand hyaluronan and an antibody to the CD44v6 isoform failed to induce neutrophil apoptosis, indicating that OX-50 antibody-mediated neutrophil apoptosis is epitope specific. This effect was specific to neutrophils since the OX-50 antibody did not induce apoptosis in other CD44-expressing cell types (lymphocytes, mesangial cells, or tubular epithelial cells). An injection of OX-50 antibody into normal rats caused a rapid and profound neutropenia, and apoptotic neutrophils could be seen in the blood by electron microscopy. Furthermore, the administration of OX-50 antibody abrogated neutrophil-dependent glomerular injury (proteinuria) on day 1 of rat antiglomerular basement membrane glomerulonephritis, whereas injury on day 10 of the disease (neutrophil independent) was largely unaffected. CONCLUSIONS: The cross-linking of specific epitopes of the CD44 molecule can rapidly induce neutrophil apoptosis in vitro and inhibit neutrophil-dependent renal injury in vivo. This finding suggests that physiological ligands of the CD44 molecule may play an important role in eliminating neutrophils from sites of inflammation, including inflammatory kidney disease.

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