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PubMed2005

Effect of cystic fibrosis exacerbations on neutrophil function.

In cystic fibrosis (CF), inflammation is caused by persistent bacterial infection from Pseudomonas aeruginosa and Burkholderia cenocepacia in the lung and is characterised by the persistent infiltration of massive numbers of neutrophils which leads to lung injury. The aim of this present study was to investigate the effects of CF exacerbations on the reactivity of peripheral blood neutrophils compared to data from a normal healthy control population. Peripheral blood neutrophils were isolated from control subjects and CF patients before and after an exacerbation of their lung disease. Isolated neutrophils were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA) and the rate of superoxide generation and elastase activity measured and compared with neutrophils from healthy age-matched controls. Neutrophils from CF patients spontaneously generated higher levels of superoxide after resolution of the exacerbation compared to control neutrophils. The stimulated generation of superoxide from control neutrophils was not significantly different from neutrophils isolated from CF patients either before or after resolution of the CF exacerbation. Neutrophils from CF patients spontaneously released more elastase than control neutrophils but released less elastase than control neutrophils in response to fMLP. The stimulated release of elastase from neutrophils was not significantly different before compared to after resolution of the exacerbation. Neutrophils from CF patients displayed a different pattern of response than those from control subjects; however, CF exacerbations did not appear to modulate neutrophil function.

Adult↗
PubMed2001

The loss of susceptibility to apoptosis in exudated tissue neutrophils is associated with their nuclear factor-kappa B activation.

Tissue neutrophils, human salivary neutrophils donated from healthy subjects and synovial fluid neutrophils collected from patients with rheumatoid arthritis were compared with circulating blood neutrophils. Concomitant treatment of circulating blood neutrophils with tumor necrosis factor-alpha (TNF-alpha) and cycloheximide induced neutrophil apoptosis, whereas the same treatment failed to induce significant apoptosis in salivary and synovial fluid neutrophils. Caspase-3 activation by TNF-alpha was observed in these tissue neutrophils, although its activity was significantly weaker than that in circulating blood neutrophils. In circulating blood neutrophils, TNF-alpha induced activation of nuclear factor-kappa B (NF-kappa B), whereas, in tissue neutrophils, NF-kappa B had been already activated without any stimulation, and no further activation was induced by the treatment with TNF-alpha. Furthermore, while pretreatment of neutrophils with an NF-kappa B inhibitor produced typical apoptotic changes in circulating blood neutrophils, this inhibitor did not produce any morphological apoptotic changes induced by TNF-alpha in tissue neutrophils. These results indicate that neutrophils undergo marked functional changes such as altered sensitivity to apoptosis-inducing stimuli in association with their exudation from blood into tissue, and that NF-kappa B activation is involved in the acquisition of resistance to TNF-alpha-induced apoptosis.

Apoptosis↗
PubMed2001

Pre-translational regulation of neutrophil L-selectin in glucocorticoid-challenged cattle.

L-selectin (CD62L) gene expression in neutrophils is commonly referred to as "constitutive" because circulating neutrophils require a constant supply of this adhesion molecule for continuous trafficking into peripheral tissues. Under normal circumstances, marginating blood neutrophils and neutrophils that become activated for migration into infected tissues rapidly shed surface CD62L that is ligated to the vascular endothelium. However, this does not shut down CD62L gene expression because these cells continue to express surface CD62L. In contrast, glucocorticoid challenges resulting from stress and hormone injections result in gradual and chronic down-regulation of CD62L on the surface of blood neutrophils. Rather than being associated with migration, this type of CD62L down-regulation associates with pronounced neutrophilia and increased susceptibility to infections. Nothing is currently known about glucocorticoid regulation of CD62L expression in neutrophils. In other cell systems, however, this steroid hormone binds to cytoplasmic glucocorticoid receptors (GR) that influence expression of glucocorticoid-responsive genes at multiple pre-translational levels. Thus, the hypothesis of the present study was that glucocorticoid challenge suppresses CD62L mRNA expression in blood neutrophils. Suppressed CD62L gene expression might help explain the chronic down-regulation of surface CD62L in neutrophils and accompanying neutrophilia. The main objectives of the study were to monitor neutrophil CD62L mRNA abundance before and during subtle and severe glucocorticoid challenges and to determine if CD62L mRNA expression correlates with degree of glucocorticoid challenge. Parturient dairy cows and dexamethasone-treated steers were used as models of subtle and severe (respectively) glucocorticoid challenges. Data presented from both models support the hypothesis and show for the first time that glucocorticoids regulate neutrophil CD62L at a pre-translational level. Results also showed that inhibited CD62L mRNA expression correlated precisely with down-regulated surface expression of CD62L on neutrophils and peak neutrophilia during severe glucocorticoid challenge. Therefore, results of this study indicate that bovine neutrophils are highly sensitive to the blood environment, displaying full capacity to alter CD62L gene expression and trafficking patterns in response to changing glucocorticoid levels. This may serve animals well when heightened inflammatory responses begin to lead to tissue damage, but may be detrimental to overall health if animals are exposed to opportunistic pathogens while stressed or undergoing glucocorticoid therapy. Although this study did not elucidate how glucocorticoids inhibit neutrophil CD62L mRNA expression, presented data implicate GR as possibly being involved because neutrophils from cattle in both models expressed GR mRNA. Further in vitro studies using purified populations of neutrophils will be required to determine if GR is directly involved in glucocorticoid regulation of CD62L gene expression and, if so, at what level.

Actins↗
PubMed1994

Decreased arachidonic acid metabolism in human platelets by autologous neutrophils: possible role of cell adhesion.

The amount of the 12-lipoxygenase and cyclo-oxygenase products, 12(S)-hydroxy-(Z,Z,E,Z)-5,8,10,14-eicosatetraenoic acid (12-HETE) and 12(S)-hydroxy-(E,E,Z)-5,8,10-heptadecatrienoic acid (HHT), in human platelets stimulated by thrombin (0.1 and 2.5 units/ml), was studied in the presence of autologous neutrophils. A decreased formation of both products was induced by unstimulated neutrophils or neutrophils challenged with N-formylmethionyl- leucyl-phenylalanine (0.1 microM) or Ca2+ ionophore A23187 (0.15 microM). The effect of neutrophils was observed only in the presence of Ca2+. 12-HETE and HHT were also produced in platelets stimulated with thrombin in the absence of Ca2+ and/or Mg2+, but their level was not altered by neutrophils. 12(S),20-Dihydroxy-(Z,Z,E,Z)-5,8,10,14-eicosatetraenoic acid (12,20-DHETE), the cytochrome P-450 product from 12-HETE in neutrophils, was hardly detected, and its level did not compensate for the decrease in 12-HETE observed after platelet and neutrophil co-incubation. 5(S),12(S)-Dihydroxy-(E,Z,E,Z)- 6,8,10,14-eicosatetraenoic acid (5(S),12(S)-DHETE), the 5-lipoxygenase product of 12-HETE in neutrophils, was never detectable. In addition, the inhibition of 12-HETE and HHT formations appeared not to be due to degradation or thrombin uptake by neutrophils, nor was the decrease observed when the two cell populations were physically separated. A monoclonal antibody against the human platelet glycoprotein GMP140 (CD62), mediating Ca(2+)-dependent platelet-neutrophil adhesion, mimicked the inhibitory effect of neutrophils in a dose-dependent fashion. Furthermore, the 12-HETE and HHT productions were not affected when platelets were stimulated in the presence of neutrophils previously incubated with sialidase, which removes the sialic acid from a sialyl Lewis(x) structure assumed to be the neutrophil receptor for platelet GMP140. We conclude that the decrease in thrombin-stimulated 12-HETE and HHT formation observed when platelets were co-incubated with autologous neutrophils might be the consequence of platelet-neutrophil adherence, presumably through platelet GMP140.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMedDate not supplied

Characterization of the neutrophil molecules identified by quinine-dependent antibodies from two patients.

BACKGROUND: Two patients with episodic pancytopenia and renal failure associated with quinine (Qn) ingestion were previously found to have Qn-dependent antibodies that reacted with red cells, platelets, and neutrophils. The purpose of these studies was to characterize the neutrophil antigens recognized by Qn-dependent antibodies from these two patients. STUDY DESIGN AND METHODS: The neutrophil molecules recognized by the Qn-dependent antibodies in the sera from the two patients were analyzed by immunoprecipitation using 125I-labeled neutrophils. Neutrophils from 13 different donors were tested. RESULTS: The Qn-dependent antibodies from Patient 1 immunoprecipitated a 60-kDa molecule on neutrophils from seven donors and an 85-kDa molecule on neutrophils from three donors. The Qn-dependent antibodies from Patient 2 reacted with a 32-kDa molecule on neutrophils from 5 donors, a 60-kDa molecule on neutrophils from 9 donors, and an 85-kDa molecule on neutrophils from 10 donors. Neutrophil-specific antigen NB1 is also located on a 60-kDa glycoprotein (GP). While the antibody in serum from Patient 1 did not show specificity for NB1, the antibody from Patient 2 detected the 60-kDa molecule on NB1-positive neutrophils from 9 of 11 donors tested and did not detect the 60-kDa molecule on NB1-negative neutrophils from 2 donors. In a monoclonal antibody immobilization of granulocyte antigens assay, the Qn-dependent antibody from both patients reacted with the 60-kDa molecule carrying NB1. The Qn-dependent antibody from a third patient, Patient 3, was previously found to react with an 85-kDa GP and the 60-kDa NB1 GP. To determine if the Qn-dependent antibodies from Patients 2 and 3 recognized the same 85-kDa GP, neutrophils were treated with serum from Patient 3 plus Qn to remove the 85-kDa GP. Then, serum from Patient 2 plus Qn no longer immunoprecipitated the 85-kDa GP. CONCLUSION: The antigens recognized by Qn-dependent neutrophil antibodies were located on molecules of 85, 60, and 32 kDa. Qn-dependent antibodies from two patients reacted with the same 85-kDa GP and those from three patients reacted with the same 60-kDa GP. The 60-kDa molecule recognized by the Qn-dependent antibodies carried the NB1 antigen.

Antibodies↗
PubMed1998

The neutrophil and preeclampsia.

Endothelial injury is common to all pathological features of preeclampsia. Neutrophil activation has been implicated in the pathophysiology of preeclampsia and requires binding and transmigration of neutrophils through the endothelium. This occurs via an interaction of endothelial adhesion molecules and surface receptors on neutrophils. Upon activation, neutrophil granules are released, the contents of which are capable of mediating vascular damage. In addition, leukotrienes are synthesized, and superoxide is generated in a respiratory burst. These products also provoke vascular damage. Neutrophil recruitment to the endothelium involves express of P-selectin and released of platelet activating factor from the endothelium. In preeclampsia there is evidence of an increase in neutrophil activation with up-regulation of neutrophil integrin expression and increased regulation of the protease elastase. Furthermore, these markers of neutrophil activation correlate with established markers of disease severity. The primary mechanism of neutrophil activation is unknown, but neutrophils in preeclampsia appear to have normal motor activity. Several potential mechanisms of neutrophil activation have been identified. They include up-regulation of cellular adhesion molecules on the endothelial surface, increased generation of tumor necrosis factor-alpha, and endothelial activation from hyperlipidemia. In additional to activation of neutrophils in preeclampsia, there may be involvement of the interleukin-6 and endothelin-1 in "priming" neutrophils for subsequent superoxide production. Activated neutrophils are likely to play a large part in the arteriopathy and endothelial damage associated with preeclampsia, but it is unclear whether neutrophil activation is the cause or the consequence of endothelial damage.

Biomarkers↗
PubMed1993

Albumin inhibits neutrophil spreading and hydrogen peroxide release by blocking the shedding of CD43 (sialophorin, leukosialin).

Spreading of neutrophils on protein-coated surfaces is a pivotal event in their ability to respond to soluble, physiologic agonists by releasing large amounts of hydrolases and oxidants. Using neutrophils plated on serum-, fibrinogen- or fibronectin-coated surfaces, we investigated the effect of human serum albumin (HSA) on spreading-dependent neutrophil responses. HSA suppressed the respiratory burst of neutrophils in response to tumor necrosis factor-alpha (TNF), complement component C5a or formylated peptide, but not phorbol myristate acetate. HSA was suppressive only if added before the onset of the respiratory burst, and suppression was reversed when HSA was removed. Likewise, HSA selectively and reversibly inhibited TNF-induced cell spreading and the associated fall in cAMP. However, HSA did not hinder TNF-induced cell adherence to the same protein-coated surfaces. We investigated cell surface sialoproteins as modulators of cell spreading and as targets for the anti-spreading action of HSA. Oxidation of the cell surface with periodate followed by reduction with 3H-borohydride and immunoblotting with specific mAbs helped identify the predominant sialoprotein on human neutrophils as CD43 (sialophorin, leukosialin). Treatment of neutrophils with C. perfringens sialidase desialylated CD43, markedly enhanced the ability of the cells to respond to TNF by spreading and undergoing a respiratory burst, and antagonized the ability of HSA to inhibit these responses. TNF-treated, adherent neutrophils shed CD43, and this was blocked by HSA, but not by ovalbumin. Exogenous neutrophil elastase removed CD43 from the neutrophil surface. HSA blocked the actions of both sialidase and elastase on CD43. In contrast, ovalbumin did not block the action of sialidase on CD43, and HSA did not inhibit the ability of sialidase to hydrolyze a synthetic substrate. These results suggested that HSA might bind CD43. In fact, the extracellular portion of CD43 bound to HSA-Sepharose, but not to ovalbumin- or glycylglycine-Sepharose. Finally, two mAbs recognizing different epitopes on CD43 mimicked HSA's inhibitory effects on neutrophil function. Thus, HSA can dissociate attachment of neutrophils from spreading. This dissociation may help neutrophils migrate along a chemotactic gradient, while decreasing their release of oxidants. CD43, a long, rigid molecule with a markedly negative charge, antagonizes neutrophil spreading. HSA appears to inhibit spreading-dependent neutrophil functions by binding to CD43 and interfering with the ability of neutrophils to shed it.

Antigens, CD↗
PubMed2003

Neutrophils pretreated with volatile anesthetics lose ability to cause cardiac dysfunction.

BACKGROUND: Volatile anesthetics can precondition the myocardium against functional depression and infarction following ischemia-reperfusion. Neutrophil activation, adherence, and release of superoxide play major roles in reperfusion injury. The authors tested the hypothesis that pretreatment of neutrophils with a volatile anesthetic, i.e., simulated preconditioning, can blunt their ability to cause cardiac dysfunction. METHODS: Studies were performed in 60 buffer-perfused and paced isolated rat hearts. Left ventricular developed pressure served as an index of myocardial contractility. Polymorphonuclear neutrophils and/or drugs were added to coronary perfusate for 10 min, followed by 30 min of recovery. Platelet-activating factor was used to stimulate neutrophils. Pretreatment of neutrophils consisted of incubation with 1.0 minimum alveolar concentration (MAC) isoflurane or sevoflurane for 15 min, followed by washout. Additional studies were performed with 0.25 MAC isoflurane. Effects of superoxide dismutase were compared to those of volatile anesthetics. Superoxide production was measured by spectrophotometry. Neutrophil adherence to coronary vascular endothelium was estimated from the difference between neutrophils administered and recovered in coronary venous effluent. RESULTS: Activated neutrophils caused marked, persistent reduction (> 50%) in left ventricular developed pressure. Isoflurane and sevoflurane at 1.0 MAC and superoxide dismutase abolished this effect. Isoflurane and sevoflurane reduced superoxide production of activated neutrophils by 29% and 33%, respectively, and completely prevented the platelet-activating factor-induced increases in neutrophil adherence. Isoflurane at 0.25 MAC blunted, but did not abolish, the neutrophil-induced decreases in left ventricular developed pressure. CONCLUSION: Neutrophils pretreated with 1.0 MAC isoflurane or sevoflurane lost their ability to cause cardiac dysfunction, while those pretreated with a concentration of isoflurane as low as 0.25 MAC were partially inhibited. This action of the volatile anesthetics was associated with reductions in superoxide production and neutrophil adherence to the coronary vascular endothelium. Our findings suggest that inhibitory actions on neutrophil activation and neutrophil-endothelium interaction may contribute to the preconditioning effects of volatile anesthetics observed in vivo during myocardial ischemia-reperfusion.

Anesthetics, Inhalation↗
PubMed2000

Neutrophil adhesion to vascular prosthetic surfaces triggers nonapoptotic cell death.

OBJECTIVE: To test the hypothesis that neutrophil adhesion to expanded polytetrafluoroethylene (ePTFE) and Dacron triggers cell death. SUMMARY BACKGROUND DATA: Vascular prosthetic infections are intransigent clinical dilemmas associated with excessive rates of death and complications. Impaired neutrophil function has been implicated in the infection of implanted cardiovascular devices. ePTFE and Dacron are potent neutrophil stimuli able to elicit activation responses such as reactive oxygen species production independent of exogenous/soluble agonists. Reactive oxygen species that are released into the medium when neutrophils are challenged by soluble agonists are known to cause self-destruction. The authors therefore sought to examine whether neutrophil adhesion to prosthetic graft materials decreases neutrophil viability by means of reactive oxygen species production. METHODS: Neutrophils were adhered to surfaces for up to 6 hours. Cell viability was monitored with propidium iodide staining and lactate dehydrogenase release. RESULTS: Within 6 hours of adhesion to ePTFE and Dacron, respectively, 59% +/- 11% and 44% +/- 5% (n = 7) of the neutrophils were stained by propidium iodide. Indistinguishable results were obtained with plasma-coated ePTFE and Dacron. In contrast, less than 2% of the neutrophils adherent to fibrinogen-, immunoglobin-, or fetal bovine serum-coated polystyrene surfaces for 6 hours were positive for propidium iodide. The increase in membrane permeability to propidium iodide was accompanied by a two- to threefold increase in lactate dehydrogenase release. Pretreatment of neutrophils with N-acetyl-L-cysteine, cytochalasin D, or cyclosporin A significantly reduced the number of propidium iodide-positive ePTFE and Dacron adherent neutrophils. CONCLUSIONS: Neutrophil adhesion to ePTFE and Dacron triggers a rapid nonapoptotic cell death. The effect of ePTFE and Dacron on neutrophil viability appears to be caused by reactive oxygen species production. The premature death of graft-adherent neutrophils provides a novel explanation of the defect in neutrophil bacterial killing associated with vascular prosthetic grafts.

Blood Vessel Prosthesis↗
PubMed1999

Circulating granulocyte macrophage colony-stimulating factor in plasma of patients with the systemic inflammatory response syndrome delays neutrophil apoptosis through inhibition of spontaneous reactive oxygen species generation.

In the normal resolution of an acute inflammatory response apoptosis of neutrophils is essential to maintain immune homeostasis and limit inappropriate host tissue damage by decreasing neutrophil tissue load, function, and release of phlogistic reactive oxygen species and proteases. The systemic inflammatory response syndrome (SIRS), a massive pro-inflammatory immune state, is associated with delayed neutrophil apoptosis, however, the systemic circulating factors and intracellular signal transduction pathways important in regulating neutrophil apoptosis in SIRS are poorly described. Neutrophils isolated from patients with SIRS on admission to the intensive care unit showed significantly (p<.01) delayed spontaneous neutrophil apoptosis compared with healthy neutrophils as quantified using annexin V-FITC and terminal deoxyuridine triphosphate (dUTD) nick end labeling (TUNEL) flow cytometry methods. Plasma from SIRS patients markedly (41.5+/-7.2%, p<.01) inhibited apoptosis of healthy neutrophils compared with controls (69.7+/-4.8%) indicating the presence of soluble circulating factors that can modify the expression of neutrophil apoptosis. Various pro-inflammatory (IL-6, granulocyte macrophage colony-simulating factor, interleukin (IL)-1beta, tumor necrosis factor-alpha) mediators, known to modulate neutrophil apoptosis in vitro, were elevated in the plasma of our cohort of SIRS patients compared with controls. However, the anti-apoptotic effect of SIRS plasma was specifically attenuated (75.5%, p<.01) by neutralizing SIRS plasma of granulocyte macrophage-colony-stimulating factor, but not IL-6, IL-1beta, tumor necrosis factor-alpha. Although the anti-inflammatory cytokine IL-10 was elevated in SIRS plasma (median level 7.2 pg/mL), further boosting SIRS plasma with recombinant human IL-10 (10 ng/mL, levels found in septic shock patients) significantly countered (63.8%, p<.01) the inhibitory effect of SIRS plasma on neutrophil apoptosis. Suppression of neutrophil apoptosis was concomitant with delayed spontaneous elevation of reactive oxygen species, quantified as peroxide production, and reversed by addition of neutralizing antibodies to GM-CSF, and recombinant human IL-10 to SIRS plasma. These results identify circulating GM-CSF as a significant inhibitor of neutrophil apoptosis in patients with SIRS, and that this effect can be countered by boosting SIRS plasma with IL-10. GM-CSF and IL-10 appear to modulate neutrophil apoptosis by altering reactive oxygen species generation in neutrophils.

Adolescent↗
PubMed2001

Ex vivo fluorescence microscopy provides simple and accurate assessment of neutrophil-endothelial adhesion in the rat lung.

Neutrophil adhesion to the pulmonary endothelium is prerequisite to neutrophil transmigration and activation, both of which may lead to lung injury. A simple method to evaluate neutrophil adherence in the lung would be useful for developing new strategies for neutrophil-mediated lung injury. The purpose was to establish a simple method to evaluate neutrophil adhesion in the lung using ex vivo fluorescence microscopy. Rats were anesthetized, and the right jugular veins were catheterized. Neutrophils were isolated from another set of rats and labeled with 5,(6)-carboxyfluorescein diacetate. Animals were killed 120 s after a 1 x 10(6) labeled neutrophil injection. The pulmonary labeled neutrophil number was counted under a fluorescence microscope. In the first experiment, rats were given 0, 20, 200, or 2000 microg/kg lipopolysaccharide (LPS) i.p. At 4 h after challenge, the pulmonary labeled neutrophil number was determined. Kinetic studies were also performed at 0, 1, 4, and 8 h after 200 microg/kg LPS. Finally, anti-ICAM-1 Ab was injected i.v. before LPS 200 microg/kg, and the labeled neutrophil number in the lung was determined at 4 h. The number of pulmonary labeled neutrophils was higher after LPS 200 or 2000 microg/kg than after the other doses. The pulmonary labeled neutrophil number was increased at 4 h compared with the other time points. ICAM-1 blocking normalized the pulmonary labeled neutrophil number in the LPS group. In conclusion, our method seems to reflect ICAM-1-mediated neutrophil adherence to the endothelium in the present setting. This simple technique may be useful for evaluating neutrophil adhesion.

Animals↗
PubMed1999

Induction of CD18-mediated passage of neutrophils by Pasteurella haemolytica in pulmonary bronchi and bronchioles.

Pasteurella haemolytica is an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released by P. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs during P. haemolytica pneumonia are poorly characterized. To determine whether the CD18 family (beta2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected with P. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated with P. haemolytica A1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P < 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces.

Animals↗
PubMed1992

Downregulation of blood and bone marrow neutrophils decreases expression of acute lung injury in sheep.

We have shown that infusion of zymosan-activated plasma (ZAP) in sheep causes acute lung injury and downregulates peripheral blood neutrophils in that elicited superoxide release is reduced for at least 24 h after the infusion. The present study was designed to test the following hypotheses: 1) peripheral blood neutrophils are representative of neutrophils marginated in the pulmonary circulation, 2) blood neutrophils are downregulated because neutrophils developing in bone marrow are similarly affected, and 3) downregulated neutrophils have a reduced capacity to produce tissue injury. In a series of experiments in 21 sheep, we showed that elicited superoxide release was similar in peripheral blood neutrophils and in marginated neutrophils washed out of the pulmonary vascular bed. Measurements of superoxide release from blood and bone marrow neutrophils collected 2-24 h after ZAP infusion revealed progressive downregulation with time and greater downregulation of superoxide release in bone marrow neutrophils compared with peripheral blood neutrophils. Finally, after downregulating peripheral blood neutrophils, subsequent infusion of ZAP in conscious sheep produced sequestration of neutrophils in the pulmonary circulation but failed to produce a sustained increase in lung lymph protein clearance. The results suggest that neutrophil downregulation, as measured in vitro, is expressed in vivo as reduced ability of neutrophils to produce tissue injury when challenged by an activating agent.

Acute Disease↗
PubMed2002

Hepatocyte growth factor is produced by blood and alveolar neutrophils in acute respiratory failure.

We tested the novel hypothesis that neutrophils in the lung or the airspaces may produce hepatocyte growth factor (HGF) in ventilated patients with acute respiratory failure. Neutrophils were purified from blood and bronchoalveolar lavage (BAL) fluid samples from 16 mechanically ventilated patients who underwent BAL for a diagnostic workup of ventilator-acquired pneumonia. Most of the patients had pneumonia (n = 11). Ten nonventilated patients served as controls. Both blood and BAL neutrophils released HGF in vitro. Basal HGF secretion by blood neutrophils from controls was 823 (666) pg x ml(-1) x 10(-7) neutrophils (median, 25th-75th percentile) and doubled to 1,730 (1,684-2,316) pg x ml(-1) x 10(-7) neutrophils (P = 0.001) with lipopolysaccharide (LPS) stimulation. Basal HGF secretion by blood neutrophils from patients was similar [956 (655-2,140) pg x ml(-1) x 10(-7) neutrophils, P = 0.4] and doubled with LPS stimulation [2,767 (2,165-3,688) pg x ml(-1) x 10(-7) neutrophils, P < 0.0001 vs. controls]. Alveolar neutrophils released HGF in vitro [653 (397-1,209) pg x ml(-1) x 10(-7) neutrophils]. LPS stimulation did not significantly increase the HGF release from alveolar neutrophils [762 (434-1,305) pg x ml(-1) x 10(-7) neutrophils]. BAL HGF positively correlated with the BAL neutrophil count (P = 0.01, R = 0.58). We conclude that blood and alveolar neutrophils from patients with acute respiratory failure can produce HGF, a mitogenic factor that may enhance the alveolar repair process.

Acute Disease↗
PubMed2003

Distinct cytokine production by lung and blood neutrophils from children with cystic fibrosis.

Inflammation plays a critical role in lung disease progression in cystic fibrosis (CF). This inflammatory process is dominated by a neutrophil influx in the airways. To determine whether the accumulation of neutrophils in the airways of CF patients is associated with an altered function, we analyzed the capacity of neutrophils isolated from the lung compartment and the blood to release the major neutrophil pro- and anti-inflammatory cytokines IL-8 and IL-1-receptor antagonist (ra) spontaneously and in the presence of LPS. Comparison of cytokine production by blood neutrophils from CF patients and from control subjects showed significantly increased IL-8 and decreased IL-1ra release by CF neutrophils. Comparison of cytokine production by airway and blood neutrophils from CF patients also documented distinct profiles: the spontaneous release of IL-8 and IL-1ra by airway neutrophils was significantly higher than that from blood neutrophils. Culture in the presence of LPS failed to further enhance cytokine production. Analysis of the effect of dexamethasone confirmed the difference in the responsiveness of lung and blood neutrophils in CF. Used at a concentration effective in reducing IL-8 production by blood neutrophils, dexamethasone (10(-6) M) was unable to repress secretion of IL-8 by airway neutrophils. In addition, comparison of cytokine production by airway neutrophils from children with CF and children with dyskinetic cilia syndrome also documented distinct profiles of secretion. These results are consistent with a dysregulated cytokine production by lung and blood neutrophils in CF. They provide support to the hypothesis that not only the CF genotype but also the local environment may modify the functional properties of the neutrophils.

Adolescent↗
PubMed1991

Pentoxifylline reduces injury to isolated lungs perfused with human neutrophils.

Neutrophils and neutrophil-derived oxidants have been implicated in the development of acute lung injury such as that seen in the adult respiratory distress syndrome (ARDS), in bronchopulmonary dysplasia (BPD), and in animal models of lung injury, including the isolated perfused lung. Both neutrophil-derived oxidant production and retention of neutrophils in the lung are required for injury in this model. Pentoxifylline can reduce lung injury from sepsis in the guinea pig and endotoxin-induced neutrophil sequestration and lung injury in the dog. It is also known to increase neutrophil deformability, which may affect retention in the pulmonary microvasculature. We evaluated neutrophil oxidant production, retention in isolated lungs, and neutrophil-mediated acute lung injury after phorbol myristate acetate (PMA) in the presence of pentoxifylline. Pentoxifylline (2 mM) significantly reduced superoxide anion and hydrogen peroxide production in vitro from PMA-stimulated neutrophils when pentoxifylline was directly added to the incubation mixtures, but not when neutrophils were preincubated with the agent. Pentoxifylline did not reduce retention of neutrophils in isolated lungs as determined by infusion of 111In-labeled neutrophils and gamma counting. Pentoxifylline prevented increases in total lung weight, lung-to-body-weight ratio, and perfusate thromboxane concentrations when it was present in perfusate buffer, whether or not neutrophils were preincubated in pentoxifylline prior to infusion into the lung. Pentoxifylline did not reduce injury to lungs perfused with glucose and glucose oxidase. We conclude that pentoxifylline reduces neutrophil oxidant production and neutrophil-dependent lung injury.

Animals↗
PubMed1998

Exogenous nitric oxide enhances neutrophil cell death and DNA fragmentation.

Inhaled nitric oxide (NO) is an important new therapeutic agent used to treat pulmonary arterial hypertension in a variety of disease states. However, the effects of NO on cells in the lung are uncertain. Previously, we have shown that NO gas depresses neutrophil oxidative cell function and increases neutrophil cell death. The purpose of this in vitro study was to determine the mechanism of neutrophil death. We hypothesized that NO hastened cell death by inducing apoptosis. To mimic the clinical environment of patients with respiratory failure, we also studied the effects of hyperoxia on neutrophil cell viability and apoptosis. Isolated human neutrophils were exposed to 80% O2 (O2), NO at 20 ppm in room air (NO/RA), 20 ppm NO blended with 80% O2 (NO/O2), or RA alone (control) for 2 to 24 h. Experiments were repeated with NO concentrations of 5 and 50 ppm and with 20 ppm in the presence of superoxide dismutase (SOD). Neutrophils were also incubated in the absence or presence of neutrophil stimulant fMLP (10 nM). Neutrophil cell viability was measured by fluorescence viability/cytotoxicity assay. Neutrophil apoptosis was assessed by cell death detection ELISA for histone-associated DNA fragments, TdT transferase-mediated fluorescence-labeled dUTP nick end labeling (TUNEL) assay, and DNA fragmentation gel electrophoresis. NO/O2-exposed neutrophils showed decreased viability at 2 h (31.7 +/- 3.7%, mean % viability +/- SD) compared with control (94.7 +/- 4.7%), O2 (75.6 +/- 9.3%), and NO/RA (62.8 +/- 14.9%; P < 0.05 by ANOVA; n = 9). Although control neutrophils demonstrated marked apoptosis at 24 h, there was no significant apoptosis at 2, 4, or 6 h (P < 0.001 by Kruskal-Wallis, n = 20) as assessed by ELISA and TUNEL assays. When compared with RA controls at 2 h, neutrophils exposed to NO/O2 showed significantly more apoptosis (292% of control, range: 106 to 2,488%, P < 0.001 by ANOVA and Kruskal-Wallis) but not with exposure to NO/RA or O2 alone. These findings were confirmed by TUNEL assay (n = 4, P < 0.05). NO/ RA and NO/O2-exposed neutrophils demonstrated both evidence of necrosis and enhanced DNA fragmentation at 2 h by gel electrophoresis (n = 2). Fifty parts per million NO produced similar findings, but exposure to 5 ppm NO did not induce significant DNA fragmentation. Coincubation with SOD inhibited NO/ O2-associated apoptosis, suggesting peroxynitrite contributed to cell death. Stimulation with fMLP did not alter apoptosis induced in neutrophils exposed to NO/RA or NO/O2. We conclude that exogenous NO gas, at clinically relevant concentrations under hyperoxic conditions, induces cell death in neutrophils in part by enhancing DNA fragmentation.

Apoptosis↗
PubMed1989

Human neutrophils contain and bind high molecular weight kininogen.

Because plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK) circulates complexed with high molecular weight kininogen (HMWK), we determined whether HMWK could mediate kallikrein's association with neutrophils. HMWK antigen (237 +/- 61 ng HMWK/10(8) neutrophils) was present in lysates of washed human neutrophils. Little if any plasma HMWK was tightly bound and nonexchangeable with the neutrophil surface. Human neutrophils were found to possess surface membrane-binding sites for HMWK but no internalization was detected at 37 degrees C. 125I-HMWK binding to neutrophils was dependent upon Zn2+. Binding of 125I-HMWK to neutrophils was specific and 90% reversible. 125I-HMWK binding to neutrophils was saturable with an apparent Kd of 9-18 nM and 40,000-70,000 sites per cell. Upon binding to neutrophils, 125I-HMWK was proteolyzed by human neutrophil elastase (HNE) into lower relative molecular mass derivatives. Furthermore, HMWK found in neutrophils also served as a cofactor for HNE secretion because neutrophils deficient in HMWK have reduced HNE secretion when stimulated in plasma deficient in HMWK or with purified kallikrein. These studies indicate that human neutrophils contain a binding site for HMWK that could serve to localize plasma or neutrophil HMWK on their surface to possibly serve as a receptor for kallikrein and to participate in HNE secretion by this enzyme.

Antigens↗