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PubMed1998

Fas ligand (gld)- and Fas (lpr)-deficient mice do not show alterations in the extravasation or apoptosis of inflammatory neutrophils.

Apoptosis of neutrophils plays a critical role in the resolution of acute inflammation. Neutrophils from human peripheral blood express Fas (CD95) and are sensitive to Fas ligand (FasL)/Fas-mediated apoptosis. Mice carrying spontaneous mutations in the genes for fas ligand (B6/gld) or fas (B6/lpr) were used to assess the role of FasL/Fas in the kinetics and magnitude of neutrophil extravasation to the thioglycolate (TG)-inflamed peritoneum and in the spontaneous apoptosis of TG-elicited neutrophils. The results showed that TG-elicited neutrophils (defined by flow cytometry as GR-1/Ly-6G(hi) cells) from normal (B6) and B6/gld mice, but not from the Fas-deficient B6/lpr mice, express high levels of Fas. The TG-elicited neutrophil response began at 2 h, peaked at 4 h, and subsided by 24-48 h after TG administration in all three strains. However, the response was more prolonged in B6 mice, such that B6/gld and B6/lpr mice had fewer neutrophils at 6 h after TG administration than did B6 mice. Further studies showed that 4 h TG-elicited neutrophils from B6, B6/gld and B6/lpr mice undergo apoptosis in vitro at similar rates (as assessed through flow cytometry by the decrease in forward angle light-scatter and externalization of phosphatidylserine (PS; as detected by Annexin V-FITC) that occur as neutrophils undergo apoptosis). Fas expression was down-regulated on apoptotic neutrophils in conjunction with maximal PS externalization and decreased forward angle light-scatter. Collectively, these findings suggest that FasL/Fas-mediated apoptosis is not essential in regulating the lifespan of neutrophils during an acute inflammatory response. The abbreviated inflammatory response observed in FasL/Fas-deficient mice is likely to be a secondary effect of the gld/lpr autoimmune/lymphoproliferative syndrome, and not a direct effect of FasL/Fas on the ability of inflammatory neutrophils to undergo apoptosis.

Animals↗
PubMed1997

Absence of the IL-7 receptor component CDw127 indicates that gamma(c) expression alone is insufficient for IL-7 to modulate human neutrophil responses.

It has been proposed that neutrophils are targets for interleukin-7 (IL-7) because this cytokine was found to increase the number of murine immature neutrophils in vivo. In addition, some nonhuman myeloid cell lines were shown to express the IL-7 receptor (IL-7R). Moreover, it was recently discovered that human neutrophils constitutively express the common gamma chain (gamma(c)), known to be a component of not only IL-7R, but also IL-2R, IL-4R, IL-9R, and IL-15R. Among these, we have recently observed that IL-4 and IL-15 are neutrophil agonists. All of the above observations prompted us to study IL-7-human neutrophil interactions. In this study, we investigated potential effects of IL-7 on a range of neutrophil responses. Although we were able to confirm the presence of the gamma(c) component on human neutrophils, we report, for the first time, that these cells lack the CDw127 component of IL-7R. When studying potential modulatory effects of IL-7 on human neutrophils, we found that IL-7 does not induce respiratory burst, phagocytosis, or cytoskeletal functions and does not alter gene expression. Positive controls were included in each assay and the expected results were obtained. In addition, in contrast to IL-4 and IL-15, we found that neutrophil apoptosis is not modulated by IL-7, while granulocyte-macrophage colony-stimulating factor, used here as control, strongly delayed this process as expected. We conclude that the sole expression of gamma(c) on human neutrophils is insufficient to modulate neutrophil responses with respect to the studied functions. Therefore, it cannot be proposed, based on studies performed with nonhuman cells or cell lines, that human neutrophils are targets for IL-7.

Animals↗
PubMed1993

The mechanism of hepatic cellular injury in sepsis: an in vitro study of the implications of cytokines and neutrophils in its pathogenesis.

Neutrophil-mediated injury to hepatocytes was evaluated in vitro. A new in vitro coculture system of neutrophils and a human hepatoblastoma cell line (HuH-6), instead of normal hepatocytes, was established. Recombinant human tumor necrosis factor (TNF) activated neutrophils to release neutrophil elastase and showed the significant cytotoxicity for HuH-6 cells, which was determined by measuring the release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), from HuH-6 cells. The concentration of neutrophil elastase from zymosan-primed/TNF (1.0 ng/ml)-stimulated neutrophils cocultured with HuH-6 cells reached to the level of 1.59 +/- 0.18 micrograms/10(6) cells in 24 hr. The release of LDH from HuH-6 cells in this coculture system was 84.8 +/- 17.8 units/liter after 24 hr incubation. Purified human neutrophil elastase also increased LDH release from HuH-6 cells. When HuH-6 cells were cocultured with zymosan-primed/TNF (1.0 ng/ml)-stimulated neutrophils, the secretion of the negative acute phase reactant (APR), alpha-fetoprotein, from HuH-6 cells was significantly decreased, and the production of the positive APR, pancreatic secretory trypsin inhibitor, was decreased in response to the stimulation of interleukin 6. Urinary trypsin inhibitor, the inhibitor of neutrophil elastase, decreased the release of LDH from HuH-6 cells cocultured with stimulated neutrophils, while superoxide scavenger did not. These results show that human neutrophils activated by TNF injure hepatocytes, thus causing hepatic dysfunction, through the release of neutrophil elastase.

Culture Media, Conditioned↗
PubMed1998

Nitric oxide donor decreases neutrophil adhesion in both lung and peritoneum during peritonitis.

BACKGROUND: As nitric oxide (NO) is an antiadhesive molecule, exogenous NO may modulate neutrophil adhesion in organs. This study was designed to examine the effects of the NO donor SNAP (S-nitroso-acetyl penicillamine) on neutrophil adhesion at the inflammatory site and in remote organs, in peritonitis using a fluorescent microscopic method. MATERIALS AND METHODS: In experiment 1, rats (n = 12) were given saline or 10 micrograms/kg of SNAP intravenously followed by continuous infusion of saline, or of 2, 20, or 200 micrograms/kg/h SNAP until sacrifice. Ten minutes after injection of saline or SNAP, 10(7) Escherichia coli were injected into the peritoneal cavity. Five hours after challenge, 10(6) fluorescein-labeled neutrophils were infused. Peritoneal samples, lungs, liver, and kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. In experiment 2, rats (n = 25) were treated with saline or 10 micrograms/kg of SNAP intravenously and infused with saline or 20 micrograms/kg/h SNAP; E. coli was injected as in experiment 1. Before or 5 h after challenge, hemodynamic data were obtained. Then, labeled neutrophils were infused for counting of neutrophil numbers in organs. Arterial blood gas data and the circulating neutrophil number were also determined. RESULTS: Experiment 1. Twenty and 200 micrograms/kg/h SNAP infusions tended to reduce labeled neutrophil numbers in lungs, while all three SNAP doses decreased the peritoneal labeled neutrophil numbers. RESULTS: Experiment 2. Five hours after bacterial injection, SNAP infusion simultaneously decreased both pulmonary and peritoneal labeled neutrophil numbers. SNAP had no effect on hemodynamic and blood gas data, or on circulating neutrophil numbers. CONCLUSION: NO donors may be useful for preventing neutrophil-associated lung injury, but should be used with caution in light of the possible adverse effects on host defense in the peritoneal cavity.

Animals↗
PubMed2002

The neutrophil as a mediator of myocardial ischemia-reperfusion injury: time to move on.

Granulocytes, especially neutrophils, are recruited in myocardium during the evolution of acute myocardial infarction. Because the neutrophil reaction is most intense during reperfusion and because these cells are a rich source of toxic oxidant species and proteolytic enzymes, it has become a widely held view that neutrophils are an important mechanism of myocardial injury extension during reperfusion. However, on close examination the evidence underlying this contention is equivocal. The basic experimental situation can be summarised thus. (1) All forms of reperfusion injury (i.e., cytotoxic or lethal cell injury, myocardial stunning, endothelial dysfunction, and reperfusion-induced arrhythmias) can be observed in neutrophil-free conditions. (2) "Anti-neutrophil" interventions (e.g., anti-inflammatory drugs, adenosine, anti-neutrophil antisera, leukocyte filters and inhibitors of the various pathways of neutrophil adhesion) do not consistently prevent reperfusion injury and they certainly do not consistently limit infarct size. (3) The time course of neutrophil accumulation in post-ischaemic myocardium may be different to the time course of injury. (4) Despite more than two decades of research, no double-blind, randomised controlled clinical trial assessing an anti-neutrophil therapy in myocardial infarction has yet reported a positive benefit that is attributable to inhibition of neutrophil recruitment. The evidence weighs against a pivotal role of neutrophils as a causal factor in most forms of ischemia-reperfusion injury. An exception may be microvascular injury and capillary plugging leading to the "no-reflow" phenomenon but even here the evidence suggests that the extent of neutrophil accumulation and microvascular injury is determined by, rather than a cause of, myocyte necrosis.

Humans↗
PubMed2006

The effects of high-intensity exercise on skeletal muscle neutrophil myeloperoxidase in untrained and trained rats.

The primary purpose of this study was to examine the effects of high-intensity acute exercise on neutrophil infiltration in different muscle fiber types of untrained rats and to compare postexercise neutrophil accumulation in muscles of untrained and trained animals. The effect of high-intensity acute exercise on blood neutrophil degranulation reaction in trained animals was also elucidated. Neutrophil enzyme myeloperoxidase (MPO) was determined as a measure of neutrophil migration into muscles and blood neutrophil degranulation. Male albino rats were subjected to acute exercise and 5 weeks of training. The used model of intensive acute exercise consisted of 5, 15, and 25 intermittent swimming bouts with the addition of weight (8% of total body mass) for 1-min each, followed by 1.5-min rest intervals. MPO was analyzed in quadriceps muscle (white and red portion) and in soleus muscle 24 h after acute exercise. MPO content in resting blood plasma and neutrophils was determined 48-h following the completion of a training process. In addition, MPO content in the trained rats was measured immediately (in blood plasma and neutrophils) after and 24 h (in muscles) following a single-bout of exercise to exhaustion. The remaining two-third of the trained animals were exposed to a single-bout of nonstop swimming with the addition of 6% body mass until exhaustion. These animals were sacrificed immediately and 24 h after loaded swimming to analyze leukocyte count, MPO content in blood plasma and neutrophils and in muscles, respectively. About 24 h after exercise MPO concentrations in the red portion of quadriceps muscle and in soleus muscle were 4-7-fold higher as compared to the white portion of m. quadriceps. There was an association between the quantity of repetitive bouts of swimming and MPO content in the muscles. The duration of swimming to exhaustion of trained rats was 3.8-fold longer than untrained sedentary control. At rest, plasma MPO concentration was found to be 40% higher in trained rats compared to untrained controls (P < 0.05). Postexercise plasma MPO concentrations were significantly higher both in untrained (+137%; P < 0.05) and trained (+81%; P < 0.05) rats compared to resting values. At rest neutrophil MPO concentration was found to be 33% lower in trained rats compared to untrained controls (P < 0.05). There were no significant differences in muscle MPO concentrations between untrained and trained rats at rest. A single-bout of exercise to exhaustion produced a greater increase in MPO content in untrained compared to trained rats. The data suggest that postexercise neutrophil infiltration is more intensive in red fibers types compared to white fiber types. A smaller neutrophil infiltration in muscles of trained animals after exhaustive exercise suggests a protective effect of previous training to muscle injury.

Animals↗
PubMed1983

Neutrophil-assisted DNA synthesis by human lymphocytes in response to mevalonic acid; enhancement by cytochalasin B.

Mevalonic acid is capable of initiating DNA synthesis, morphologic transformation, and cell division in cultures of human peripheral blood lymphocytes. Pure populations of lymphocytes respond poorly to mevalonic acid, but their response can be enhanced by peripheral blood neutrophils. Addition of cytochalasin B (0.5-1.0 micrograms/ml), but not cytochalasin A, to mixed neutrophil-lymphocyte cultures enhances the response of lymphocytes to both Con A and mevalonate, but the increment in mevalonate-induced DNA synthesis (+343%) far exceeds the enhancement which cytochalasin B produces in the Con A response (+24%). As a consequence, the DNA synthetic response in mevalonate (10(-2) M) containing cultures averages 39% of the response to an optimal dose of Con A. The cytochalasin B-enhanced response of mixed neutrophil-lymphocyte cultures to mevalonate is abolished by prior X irradiation of the lymphocytes, or their pretreatment with mitomycin C, as well as by the addition of hydroxyurea to the cultures but is not altered by prior X irradiation or mitomycin C pretreatment of the neutrophil helper population. These experiments suggest that the enhancing effect of cytochalasin B in the response of mixed neutrophil-lymphocyte cultures to mevalonic acid results from its ability to potentiate a time-dependent conditioning effect on lymphocytes which neutrophils exert. The conditioning effect of neutrophils cannot be achieved by cell-free neutrophil lysosomal enzymes released by exocytosis, and reactive oxygen species potentially generated by neutrophils are not involved. Attempts to demonstrate the production by neutrophils of a soluble mediator induced by mevalonate in the presence of cytochalasin B were without success. In the presence of cytochalasin B, only the metabolically active R(-) enantiomeric form of mevalonate is capable of initiating DNA synthesis in mixed neutrophil-lymphocyte cultures. The cytochalasin B-potentiated response of mixed neutrophil-lymphocyte cultures to mevalonic acid is preferentially displayed in cultures containing E-rosette-negative, as opposed to E-rosette-positive, lymphocytes. These observations add to the growing knowledge about the relationship between mevalonate metabolism, DNA synthesis, and cell replication.

Cell Cycle↗
PubMed1991

Low-dose chymotrypsin treatment inhibits neutrophil migration into sites of inflammation in vivo: effects on Mac-1 and MEL-14 adhesion protein expression and function.

Antibody blocking studies in the mouse suggest that the MEL-14 antigen is involved in neutrophil-endothelial cell interactions and may be important in neutrophil extravasation to sites of inflammation in vivo. We recently showed that chemotactic factor activation causes a rapid (within minutes) shedding of a large fragment of the MEL-14 antigen from the surface of neutrophils. We report here that chymotrypsin, at low doses (0.1 units/1 x 10(6) cells), but not trypsin, elastase, or collagenase, causes an activation-independent rapid loss (greater than 90%) of the MEL-14 antigen from the surface of murine neutrophils. Under the same treatment conditions chymotrypsin has no effect on the expression of four other neutrophil surface antigens, including the Mac-1 adhesion protein. Chymotrypsin treatment has no effect on neutrophil adhesion to plastic, migration to C5a, regulation of the Mac-1 antigen, but causes a greater than 95% reduction in neutrophil binding to high endothelial venules (HEV) in peripheral lymph nodes measured in the ex vivo frozen section HEV binding assay. The level of inhibition of neutrophil adhesion to HEV was comparable to that seen with the MEL-14 antibody. This experimental system allows us for the first time to specifically examine the consequences of removing the MEL-14 antigen from the surface of neutrophils on function in vivo. We show that treatment with chymotrypsin blocks greater than 85% of the ability of neutrophils injected back into the animal to home to the inflamed peritoneum. In similar in vivo experiments the MEL-14 antibody blocks neutrophil homing by 60-70%. These results further support the importance of the MEL-14 antigen in neutrophil extravasation in vivo and indicate that chymotrypsin could be useful in examining the molecular mechanisms involved in extravasation of leukocytes into a variety of diverse tissue sites of inflammation.

Animals↗
PubMed1990

Flow cytometric study of injuries in cultured endothelial cells by neutrophils of the inherited cataract rats.

We examined the mechanism of endothelial injuries in the inherited cataract rats (ICR), which have a number of age-associated spontaneous injuries in the aortic endothelium. Cell cycle traverse rate of endothelial cells of ICR was shorter than that of Wistar rats. The rate was estimated from bromodeoxyuridine (BrdU) incorporation into cell nuclei measured periodically after BrdU pulse labeling. Next we established the method for measurement of cultured endothelial cell injury by neutrophils with flow cytometry by assessing the regeneration of injured endothelial cells. By the use of the gate analysis method, contaminated neutrophils were excluded from the analysis. Endothelial cell injury by neutrophils of Wistar rats was detectable at 1 x 10(5) neutrophils (1 neutrophil to 1 endothelial cell) when stimulated with 10 ng/ml phorbol myristate acetate (PMA). Extent of injury increased with an increasing number of neutrophils and the concentration of a stimulator, PMA. We detected endothelial cell injury by ICR neutrophils not only when they were stimulated but also in a resting condition, and ICR neutrophils yielded more injury than Wistar rat neutrophils. Number of adhered neutrophils to endothelial cells and effects of plasma or lymphocytes were the same between two strain rats. Scavengers of hydrogen peroxide and singlet oxygen inhibited the ICR neutrophil-induced endothelial cell injury. These findings indicate that an increase of generation of excited oxygen species from neutrophils, particularly of singlet oxygen, may cause injury of endothelial cells in this specific strain of rats.

Animals↗
PubMed1982

The labeling of rabbit neutrophils with [111In]oxine.

We report here the successful labeling of rabbit peripheral blood neutrophils with [111In]oxine. We found that standard techniques for preparation of rabbit neutrophils, while acceptable for maintenance of in vitro function, rendered the neutrophils ineffective for in vivo use after labeling with 111In. Specifically, rabbit neutrophils were sensitive to the use of hypotonic shock for red cell elimination, centrifugation into a button during preparation, and the presence of oxine during chemotaxis in vitro. Using a carefully modified method of neutrophil preparation and labeling, we found that 111In-labeled rabbit neutrophils retained normal in vitro function, including chemotaxis. In addition, using our method, 34% +/- 5% of labeled neutrophils were recoverable in peripheral blood 5 min after intravenous injection. The half-life of circulating radio-labeled neutrophils was 5.6 +/- 2 h. Continuous external imaging of radio-labeled neutrophils after intravenous injection showed initial lung uptake, followed by rapid clearance of radioactivity in the lungs (50% clearance in 10.5 +/- 3.3 min.) Hepatic radioactivity was maximal by 30 min after injection and thereafter slowly declined. Finally, we found that 111In-labeled rabbit neutrophils migrated to sites of artificially induced inflammation. Our findings indicate that 111In-labeled rabbit neutrophils, if prepared under optimal conditions, should provide a useful tool for investigating the fate of neutrophils in experimental inflammatory conditions in this animal.

Animals↗
PubMed1991

Primed neutrophils injure rat lung through a platelet-activating factor-dependent mechanism.

Bacterial lipopolysaccharide (LPS) promotes transient lung neutrophil sequestration. These LPS-primed neutrophils, when stimulated by an N-formyl peptide (FNLP), promote lung injury. We hypothesized that LPS-primed, FNLP-stimulated neutrophils promote lung injury through a platelet-activating factor (PAF)-dependent mechanism. Rats were pretreated with either saline or WEB2170, a PAF receptor antagonist (10 mg/kg po). One hour after pretreatment, rats were administered intraperitoneal LPS (salmonella typhimurium lipopolysaccharide, 500 micrograms/kg) followed 6 hr later by intravenous FNLP (250 micrograms/kg infused over 30 min). Two hours after the initiation of FNLP infusion, rats were sacrificed and assays were performed to measure: (1) lung neutrophil sequestration with myeloperoxidase (MPO) activity; (2) circulating neutrophil activation with nitroblue tetrazolium (NBT) staining, and (3) lung microvascular leak with 125I-albumin flux. We found that lung myeloperoxidase, circulating neutrophil NBT staining, and lung 125I-albumin flux were increased (P less than 0.05) in saline-pretreated LPS/FNLP rats, relative to control. While lung MPO remained increased (P less than 0.05) in WEB2170-pretreated LPS/FNLP rats, circulating neutrophil NBT and lung 125I-albumin flux were decreased (P less than 0.05) relative to those in saline-pretreated rats. We conclude that PAF mediates LPS/FNLP-induced neutrophil activation and lung injury, but is independent from lung neutrophil sequestration. Thus, lung neutrophil sequestration does not inevitably produce lung injury. Rather, neutrophils can accumulate in the lung without causing lung injury if neutrophil activation can be blocked.

Animals↗
PubMed1985

Role of alveolar macrophages in endotoxin-induced neutrophilic alveolitis in rats.

Although bacterial endotoxins have potent effects on blood monocytes and tissue macrophages, the role of alveolar macrophages in regulating intrapulmonary neutrophil traffic following endotoxemia has not been studied previously. We have previously reported that a single intraperitoneal injection of endotoxin from Escherichia coli serotype 055B5 causes acute lung inflammation by neutrophils (PMN) in rats. The factors which influence the migration of PMN in the lung in this model are unknown. To determine whether macrophage-derived products could play a role in directing migration, we enumerated neutrophils in histologic sections and employed electron microscopy to document the location of neutrophils in the lung in vivo following endotoxin. We also cultured the alveolar macrophages recovered by lung lavage to measure the effect of their culture supernatants on neutrophil migration in vitro. In the first 6 hr following endotoxin, and also 24 hr later, there was an increase in the number of PMN enumerated in the lung parenchyma by light microscopy. Electron microscopy showed the location of the neutrophils to be exclusively intravascular at 6 hr. By contrast, neutrophils were observed in both interstitial and bronchoalveolar spaces at 24 hr, confirming that transvascular migration was active at that time. The pulmonary macrophages which were recovered by lung lavage from groups of rats sacrificed at 4 and at 15 hr following the administration of endotoxin were assayed for the release into culture media of migration-stimulatory activity for neutrophils. Macrophages from animals sacrificed 4 hr following endotoxin released less migration-stimulating activity into media than macrophages from controls. These macrophages could be stimulated to release migration-stimulating activity into culture media at levels comparable to macrophages from controls by the addition of opsonized Zymosan to the culture media. By contrast, macrophages from animals sacrificed 15 hr after endotoxin spontaneously released more migration-stimulating activity for neutrophils than did macrophages from controls. Thus, in this model, a specific increase in the synthesis or release by alveolar macrophages of factors which stimulate the migration of neutrophils in vitro coincided with a transition from intravascular to extravascular alveolar inflammation by neutrophils in vivo. These observations are consistent with the hypothesis that pulmonary alveolar macrophages may contribute to the regulation of alveolar inflammation following endotoxemia by releasing factors which influence the migration of neutrophils.

Animals↗
PubMed1995

Conversion of xanthine dehydrogenase to xanthine oxidase in bovine carotid artery endothelial cells induced by activated neutrophils: involvement of adhesion molecules.

We have demonstrated that the endothelial cell-derived superoxide anion is deeply involved in the endothelial cell injury induced by activated neutrophils (Fujita, H., Morita, I. and Murota, S. (1994) Arch. Biochem. Biophys. 309, 62-69). To clarify the mechanism underlying the increase in the endothelial cell-derived superoxide anion induced by activated neutrophils, the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) in cultured endothelial cells isolated from bovine carotid arteries was investigated. Although the endothelial cells expressed both XD and XO activity, the XO activity of unstimulated cells comprised about 12% of the total (XD + XO) activity. When endothelial cells were exposed to neutrophils activated with phorbol 12-myristate 13-acetate (PMA), XO activity rapidly increased about 3-fold over the control. Whereas treatment of endothelial cells with PMA alone or unstimulated neutrophils alone did not increase the XO activity at all. The increase in XO activity in endothelial cells was also observed on the treatment of the cells with neutrophils activated with leukotriene B4 or thrombin. To determine whether or not proteases released from activated neutrophils are involved in the increased conversion of XD to XO in endothelial cells, the effects of the elastase specific inhibitor, ONO-5046, and protease inhibitors, such as aprotinin, gabexate mesylate and urinastatin, were examined. However, these protease inhibitors did not suppress the conversion of XD to XO induced by PMA-activated neutrophils. Moreover, the treatment of endothelial cells with purified human neutrophil elastase and H2O2 also did not affect the conversion at all. In contrast, monoclonal antibodies against CD11a and CD18 significantly inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. Moreover, tyrosine kinase inhibitors such as staurosporin and herbimysine also inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. These results indicate that the adhesion of activated neutrophils to endothelial cells via CD11a/CD18-ICAM-1 is involved in the conversion of XD to XO in endothelial cells induced by activated neutrophils.

Animals↗
PubMed2005

Neutrophil survival-enhancing activity in sputum from patients with diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) is a life-threatening airway disease in which neutrophils persistently and massively emigrating to the airways cause progressive and irreversible tissue damage. However, the pathogenesis of the airway inflammation remains unclear. The failure of non-inflammatory removal of emigrating neutrophils due to delayed apoptosis has been proposed as a mechanism by which the neutrophilic inflammation persists. Therefore, we aimed to investigate whether an activity that delays neutrophil apoptosis is present at the inflamed sites in DPB. Neutrophils isolated from normal volunteers were cultured with sputum extracts of patients with DPB, and viability and apoptosis of neutrophil was evaluated for the culture period. Neutrophils cultured with sputum extracts for 2 and 3 days showed significantly enhanced survival compared to those with medium alone. The neutrophil survival-enhancing activity in sputum extracts was heat-labile and partially, but significantly, neutralized with anti-human GM-CSF, but not with anti-human G-CSF antibody. The enhancement of neutrophil survival was associated with an inhibition of apoptosis demonstrated by cytology, TUNEL assay and DNA fragmentation analysis. These results suggest that neutrophil apoptosis is prevented by survival-enhancing factors including GM-CSF in the airways of DPB, leading to neutrophil death by necrosis that causes further recruitment and activation of neutrophils.

Adult↗
PubMed2006

Differential surface expression of CD18 and CD44 by neutrophils in bone marrow and spleen contributed to the neutrophilia in thalidomide-treated female B6C3F1 mice.

Previously, we have reported that thalidomide (Thd) can enhance neutrophil function in female B6C3F1 mice. The present study was intended to evaluate the mechanisms underlying the enhanced neutrophil responses following Thd treatment intraperitoneally (100 mg/kg) for 14 or 28 days. Treatment with Thd increased the numbers of neutrophils in the spleen, peripheral blood, bone marrow, peritoneal cavity and lungs of female B6C3F1 mice when compared to the vehicle control mice. Thd treatment for 14 days increased the percentage and the number of neutrophils in the spleen in the first 8 h (peaking at 2 h) after the last Thd treatment, and it returned to the baseline after 24 h. However, Thd treatment for 28 days increased the percentage and number of neutrophils in the spleen even at the 24-h time point after the last Thd treatment. These neutrophils were demonstrated to be functional by the myeloperoxidase activity assay. Further studies have ruled out the possibility of an increased bone marrow granulopoiesis following Thd treatment. Flow cytometric analysis of the surface expression of adhesion molecules suggested that Thd treatment for either 14 or 28 days decreased the surface expression of either CD18 or CD44 by bone marrow neutrophils. On the other hand, the surface expression of both CD18 and CD44 by splenic neutrophils was increased following Thd treatment for 28 days but not for 14 days. No effect was produced for other cell surface molecules such as CD62L and CD11a. It was possible that decreased surface expressions of CD18 and CD44 facilitated neutrophils' release from the bone marrow; increased surface expressions of CD44 and CD18 by splenic neutrophils after 28 days of Thd treatment increased their ability to remain in the periphery. Taken together, Thd treatment increased neutrophils in female B6C3F1 mice, at least partially, through differentially modulating the surface expression of CD18 and CD44 by the neutrophils in the bone marrow and spleen.

Animals↗
PubMed1999

Maternal neutrophil apoptosis in normal pregnancy, preeclampsia, and normotensive intrauterine growth restriction.

OBJECTIVE: In normal pregnancy there is both a neutrophilia and a mild neutrophil activation. In preeclampsia both direct and indirect evidence supports further marked neutrophil activation. In the pathogenesis of preeclampsia peripheral blood neutrophils may play a vital role in communicating between the preeclamptic placenta and the maternal vascular endothelium and contribute to the endothelial cell dysfunction that characterizes the maternal syndrome of preeclampsia. Preeclampsia shares many elements with the systemic inflammatory response syndrome. Neutrophils, key effectors of the systemic inflammatory response syndrome, are associated with hepatic necrosis and adult respiratory distress syndrome, both of which most commonly kill women with preeclampsia. We hypothesized that delayed neutrophil apoptosis could explain (1) the neutrophilia of normal pregnancy and (2) the differential maternal responses to the shared placental abnormality of preeclampsia and normotensive intrauterine growth restriction. STUDY DESIGN: Neutrophils were isolated (dextran 500, Ficoll [Amersham Pharmacia Biotech AB, Uppsala, Sweden], and erythrocyte lysis) from (1) case patients with preeclampsia at </=34 weeks' gestation, (2) healthy pregnant control subjects, (3) case patients with normotensive intrauterine growth restriction at </=34 weeks' gestation, and (4) nonpregnant female control subjects. Apoptosis was determined after 18 hours of incubation (with or without endotoxin or anti-Fas monoclonal antibody) by deoxyribonucleic acid profile (propidium iodide study), annexin V binding, and CD16 expression. RESULTS: Compared with propidium iodide profile values in nonpregnant women (median, 25%; range, 14%-40%) neutrophil apoptosis was significantly delayed in normal pregnancy (median, 9.5%; range, 7.6%-15%) and normotensive pregnancy with intrauterine growth restriction (median, 11%; range. 9.3%-19%) and was further delayed in preeclampsia (median, 6.9%; range, 4.1%-8. 2%; P </=.005 vs normal pregnancy and normotensive intrauterine growth restriction). All neutrophils remained sensitive to endotoxin inhibition but were resistant to anti-Fas induction of apoptosis. Spontaneous neutrophil apoptosis decreased as gestational age increased (r (2) = 0.48). CONCLUSIONS: Impaired neutrophil apoptosis may explain the neutrophilia associated with normal pregnancy. In women with preeclampsia activated neutrophils remain in the circulation, perhaps contributing to the persistence of preeclampsia after delivery. Neutrophils appear to modulate the variation in maternal response between preeclampsia and normotensive intrauterine growth restriction.

Adult↗
PubMed1999

Pump prime only aprotinin inhibits cardiopulmonary bypass-induced neutrophil CD11b up-regulation.

BACKGROUND: The expression of neutrophil integrin CD11b is up-regulated after cardiopulmonary bypass (CPB) and is the neutrophil adhesive molecule of most importance in neutrophil- endothelial adherence. This neutrophil-endothelial adherence is responsible for post-CPB neutrophil-induced reperfusion injury. Low-dose aprotinin protocols inhibit the CPB-induced neutrophil CD11b up-regulation. This investigation was undertaken to evaluate the effects of pump prime only aprotinin (280 mg) on the CPB-induced up-regulation of this neutrophil integrin. METHODS: Twenty-two patients scheduled for elective myocardial revascularization were randomized into two groups: (1) control (n = 12), or (2) pump prime only aprotinin (280 mg) (n = 10). Neutrophils were isolated at baseline, 50 minutes of CPB, and 30 minutes after CPB and neutrophil CD11b expression was measured. RESULTS: The control group demonstrated a significant (p < 0.05) increase in neutrophil CD11b immunofluorescent staining at 50 minutes of CPB and at 30 minutes after CPB when compared to same group baseline and to the pump prime only aprotinin group at similar time intervals. CONCLUSIONS: These results indicate that pump prime only aprotinin modulates the CPB-induced up-regulation of neutrophil CD11b integrin, an important indicator of the systemic inflammatory response to CPB. In addition to blunting of the CPB-induced up-regulation of this neutrophil integrin expression, this pump prime only dose of aprotinin is also reported to be effective at reducing post-CPB bleeding and transfusion requirements. This salutary effect of pump prime only aprotinin suggests that such low-dose regimens can be both therapeutically effective and cost effective.

Aged↗
PubMed2002

Expression of estrogen receptor subtypes and neuronal nitric oxide synthase in neutrophils from women and men: regulation by estrogen.

OBJECTIVES: (a) To identify the subtype of estrogen receptor (ER) expressed in neutrophils from premenopausal women and in neutrophils from men under different estrogen conditions and (b) to analyze the association between the modifications in the expression of ER subtypes and neuronal nitric oxide synthase (nNOS) expression induced by estrogen. METHODS: Neutrophils were isolated from pre-menopausal women during different stages of the menstrual cycle and from ten men for in vitro estrogen incubations. RESULTS: Neutrophils from premenopausal women expressed both ERalpha and ERbeta subtypes which were increased in the ovulatory phase of the menstrual cycle. Neutrophils derived from men also expressed ERalpha and ERbeta but only ERalpha expression was enhanced by in vitro incubation with 17beta-estradiol (10(-8) mol/l). In vitro incubation of neutrophils from women with 17beta-estradiol enhanced expression of both ER-alpha and ER-beta subtypes. nNOS protein was overexpressed in neutrophils from premenopausal women during the ovulatory phase. 17beta-Estradiol (10(-8) mol/l) also increased nNOS protein expression in neutrophils derived from men. Mithramycin A (10(-6) mol/l) and curcumin (10(-6) mol/l), prevented the upregulation of nNOS and ERalpha in neutrophils derived from men, suggesting the involvement of AP-1 and Sp-1 transcription factors. CONCLUSIONS: Although the in vivo levels of circulating estrogen concentrations seem to be associated with overexpression of both ERalpha and ERbeta in neutrophils from premenopausal women, which was further confirmed by the in vitro experiments with neutrophils from women, in vitro incubation of neutrophils from men with 17beta-estradiol only increased ERalpha protein expression which was associated with enhanced expression of nNOS protein.

Adult↗