Neutrophil-mediated Endothelial Cell Injury Research Articles

The Role of Neutrophil Activation in the Pathogenesis of Kawasaki Disease

In the acute phase of Kawasaki disease (KD), there is an increase in the number of circulating neutrophils, including toxic neutrophils, which are caused by a morphological change. The functions of the neutrophils are also activated, leading to the excessive production of reactive oxygen species and elastase, which may induce endothelial cell (EC) injury. Histological findings demonstrate that numerous neutrophils infiltrate into coronary artery lesions (CALs) during the early phase of KD. Thus, it is suggested that neutrophil-mediated EC injury may be involved in the formation of CALs in KD patients. In this review, we focus on the role of neutrophil activation in the pathogenesis of KD vasculitis and discuss the methods of treatment for neutrophil-mediated EC injury.

Generation of Reactive Oxygen Species by Neutrophils and Endothelial Cell Injury in Normal and Preeclamptic Pregnancies

The aim of this study was to investigate the role of neutrophil-derived reactive oxygen species on endothelial cell dysfunction in preeclampsia. We first assessed the correlation between nitrite and superoxide anion production in normal nonpregnant (n=10), normal pregnant (n=15), and preeclamptic women (n=12). We then examined neutrophil-mediated oxygen radical damage to human umbilical vein endothelial cells in vitro. Neutrophil superoxide release was measured by cytochrome C reduction; nitrite release was measured by the modified Griess reaction, and endothelial cell injury was measured by 51Cr release. N-formyl-methionyl-leucyl-phenylalanine-stimulated superoxide release by neutrophils was significantly increased in women with preeclampsia compared with the other 2 groups. Nitrite release by neutrophils was significantly decreased in preeclampsia compared with normal pregnancy. When neutrophils were pretreated with superoxide dismutase, nitrite release by neutrophils did not differ between normal pregnancy and preeclampsia, suggesting that excess superoxide anion in preeclampsia could reduce bioavailability of nitric oxide through neutrophil autocrine function. Neutrophil-mediated endothelial cell injury was significantly greater in women with preeclampsia than in the other 2 groups. Hydrogen peroxide was important in neutrophil-mediated endothelial cell injury in preeclampsia as catalase inhibited endothelial cell injury. When neutrophils were pretreated with NG-nitro-L-arginine methyl ester, neutrophil-mediated endothelial cell injury in preeclampsia was decreased, indicating a role for peroxynitrite formation as a mechanism of endothelial cell injury. In conclusion, the modulation of neutrophils causing superoxide production to dominate over nitrite release provides a reasonable explanation for endothelial cell dysfunction in preeclampsia.

Trauma/hemorrhagic shock mesenteric lymph upregulates adhesion molecule expression and IL-6 production in human umbilical vein endothelial cells.

Trauma/hemorrhagic shock (T/HS) is associated with significant lung injury, which is mainly due to an inflammatory process, resulting from the local activation and subsequent interaction of endothelial cells and leukocytes. Adhesion molecules expressed by both cell types play a crucial role in the process of neutrophil-mediated endothelial cell injury. We have previously shown that mesenteric lymph duct ligation prevents T/HS-induced lung leukocyte infiltration and endothelial injury, suggesting that inflammatory factors originating from the gut and carried in the lymph are responsible for the lung injury observed following T/HS. Based on these observations, we hypothesized that inflammatory substances in T/HS lymph trigger lung injury by a mechanism involving the upregulation of adhesion molecules. To test this hypothesis, we examined whether T/HS mesenteric lymph induces the expression of E-selectin, P-selectin, and intracellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs). Furthermore, because the cytokine IL-6 is an important component of the endothelial inflammatory process, we investigated how T/HS lymph affects the production of IL-6 by HUVECs. Mesenteric lymph from T/HS rats increased both E- and P-selectin, as well as ICAM-1 expression on HUVECS, as compared to trauma/sham shock (T/SS) lymph or medium only groups. However, T/HS lymph failed to induce the shedding of E-selectin. In HUVECs treated with T/HS lymph, IL-6 concentrations were higher than HUVECs treated with T/SS lymph. These findings suggest that mesenteric lymph produced after hemorrhagic shock potentiates lung injury by the upregulation of endothelial cell adhesion molecule expression and IL-6 production.

Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury

To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil-mediated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbilical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose-dependently by urinary trypsin inhibitor (ulinastatin, UTI) and ONO-5046, which have the ability to inactivate neutrophil elastase, but not by gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban, which have no ability to inactivate neutrophil elastase. In addition, when UTI and ONO-5046 were added to the tumor necrosis factor alpha-primed neutrophils alone, they showed a dose-dependent inhibition of the intracellular elastase activity, but the other SPI did not, for either flow cytometry or confocal microscopy. Therefore, UTI and ONO-5046 may protect EC against the neutrophil-mediated injury not only by inactivating the extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils and suppressing the production and secretion of activated elastase from them.

Enhanced polymorphonuclear neutrophil-mediated endothelial cell injury and its relation to high surgical mortality rate in cirrhotic patients

OBJECTIVE: A high incidence of complications has been documented in patients with cirrhosis after operations. Recently, polymorphonuclear neutrophils (PMN) have been revealed to have the capacity to injure vascular endothelium and to cause organ damage. Furthermore, the altered function of PMN has been shown in patients with cirrhosis. However, there are few reports investigating the interaction between PMN and endothelial cells and its relation to a high incidence of postoperative complications in cirrhosis. The aim of this study was to evaluate PMN-mediated endothelial cell injury in patients with cirrhosis. METHODS: Patients were divided into two groups: those who had normal liver with metastatic liver carcinoma and those who had cirrhosis with hepatocellular carcinoma. All patients in both groups underwent hepatic resection. PMN were isolated from patients before operation. Human umbilical vein endothelial cells and PMN were cocultured after addition of phorbol myristate acetate. The release of lactate dehydrogenase (LDH) and thrombomodulin in the cocultured medium and the elastase activity in PMN suspension were measured. RESULTS: The release of both LDH and thrombomodulin in the group with cirrhosis was significantly higher than in the group with normal liver. The elastase activity was similarly higher in the group with cirrhosis than in the group with normal liver. The surgical morbidity rate was remarkably higher in the group with cirrhosis (50%) than in the group with normal liver (0%). CONCLUSIONS: This study shows that PMN have an enhanced potential to cause endothelial cell injury in patients with cirrhosis. This PMN “priming” may be responsible for the occurrence of postoperative complications in patients with cirrhosis after hepatectomy.

POST-HEMORRHAGIC SHOCK MESENTERIC LYMPH IS CYTOTOXIC TO ENDOTHELIAL CELLS AND ACTIVATES NEUTROPHILS

The goal of these experiments was to test the hypothesis that after a nonlethal episode of hemorrhagic shock, factors carried in the mesenteric lymph would promote endothelial cell injury and activate neutrophils to a greater extent than portal vein plasma. Catheters were placed in the efferent lymphatic duct draining the mesenteric lymph node complex, after which male rats were subjected to sham or actual shock (30 mmHg for 90 min), and lymph was collected. Portal vein plasma was collected from the sham-shock and shocked rats at 6 h post-shock or sham-shock. When the effect of lymph or portal blood plasma was tested on endothelial cell (HUVEC) monolayer permeability, it was found that post-shock lymph, but not post-shock portal vein plasma, increased HUVEC permeability to both 10 kDa and 40 kDa permeability probes. Subsequent experiments documented that only post-shock lymph was cytotoxic to endothelial cells as manifest both by decreased trypan blue dye exclusion and the increased release of Chromium-51 from chromium-loaded endothelial cells. Furthermore post-shock lymph induced a greater increase in neutrophil superoxide formation than pre-shock lymph, pre-shock, or post-shock portal vein plasma. Lastly, neutrophil-mediated endothelial cell injury was potentiated by the presence of post-shock lymph, and the magnitude of HUVEC injury was greater in endothelial cells incubated with post-shock lymph plus neutrophils than in monolayers incubated with post-shock lymph or neutrophils alone. These results suggest that post-shock lymph is cytotoxic to endothelial cells and activates neutrophils. Since the lung is the first organ that is exposed to mesenteric lymph, lung injury after hemorrhagic shock may be mediated by factors contained in mesenteric lymph.

Interactive role of nitric oxide and superoxide anion in neutrophil-mediated endothelial cell injury.

This study addresses the interactive role of nitric oxide (NO) and reactive oxygen intermediates (ROI) by direct quantitation of NO and superoxide (O2-) in human neutrophil (PMN)-endothelial cell (EC) co-culture during PMN-mediated EC injury. The results directly demonstrate an inverse correlation between NO and ROI levels in PMN-EC co-culture, which significantly alters the PMN-EC adhesion and PMN-mediated EC killing. N-formylmethionyl-leucyl-phenylalanine (fMLF)-stimulated PMN adhesion to cytokine-treated EC was decreased (> 25%) in the presence of S-nitroso-N-penicillamine, a NO donor. NO also inhibited EC killing by stimulated PMN, suggesting its cytoprotective role. In addition, a significant decrease in NO levels was observed in the PMN-EC co-culture compared with the EC cultured alone (422.45 +/- 35.76 vs. 800.79 +/- 41.69 pmol). The reduced NO levels were restored by the addition of superoxide dismutase, a scavenger of O2-, suggesting that PMN-derived O2- is involved in the neutralization of NO in the co-culture. The results indicate an inverse correlation between NO and O2- in PMN-EC interactions and suggest the need for a critical balance between these two radicals in the regulation of PMN-mediated tissue injury.

Enhanced endothelial cell injury by activated neutrophils in patients with obstructive jaundice

Background/Aims: A high incidence of complications has been documented in patients with obstructive jaundice after operations. Recent reports have revealed that neutrophils are involved in the mechanism causing postoperative complications. However, there are few reports investigating the interaction between neutrophils and endothelial cells in obstructive jaundice. The aim of this study was to evaluate neutrophil-mediated endothelial cell injury in patients with obstructive jaundice. Methods: Patients were divided into three groups: those with normal liver, obstructive jaundice, and relief of obstructive jaundice. Neutrophils were isolated from patients individually. Human umbilical vein endothelial cells and neutrophils were co-cultured after addition of phorbol myristate acetate. The release of lactate dehydrogenase and thrombomodulin was measured in the medium. Results: Lactate dehydrogenase and thrombomodulin were released in the co-culture medium, and Eglin C, which is an elastase inhibitor, reduced the release of lactate dehydrogenase and thrombomodulin in a dose-dependent manner. The release of lactate dehydrogenase and thrombomodulin in the group with relief of obstructive jaundice was significantly higher than in the group with normal liver. There was no significant difference between the group with obstructive jaundice and the group with normal liver. The elastase activity in neutrophil suspension was similarly higher in the group with relief of obstructive jaundice than in the group with normal liver. Conclusions: This study suggests that neutrophils in patients with obstructive jaundice are not activated before its relief. After relief of obstructive jaundice, neutrophils are strongly primed and have the potential to cause endothelial cell injury. The neutrophil “priming” in patients with obstructive jaundice may be associated wtih the frequent occurrence of postoperative complications.

In vitro effect of ascorbic acid on neutrophil-endothelial cell interaction.

The effect of different concentrations (0.06, 0.6 and 6.0 mmol/L) of ascorbic acid on neutrophil-endothelial interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. The aim of the study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury, the influence of ascorbic acid on adherence and endothelial cell injury have been investigated. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 6 mmol/L ascorbic acid during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 6 mmol/L (p < 0.0005). The inhibition of neutrophil adherence to endothelial cells was correlated with a diminished neutrophil-mediated endothelial cell injury during incubation with 6 mmol/L ascorbic acid (p < 0.0005). The present results indicate that ascorbic acid might exert a protective effect on neutrophil-mediated endothelial cell injury by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Moreover, the current investigation points to probable protective effect of ascorbic acid on oxidant-mediated cell damage in diseases (e.g., Adult Respiratory Distress Syndrome).

Role of platelet-activating factor in the development of endothelial dysfunction in hemorrhagic hypotension and retransfusion

Platelet-activating factor (PAF), an important mediator of ischemic and shock states, has been shown to prime direct and neutrophil-mediated endothelial cell injury. In the present study we investigated therefore whether PAF is involved in the development of dysfunction of the cerebrovascular endothelium in hemorrhagic hypotension and retransfusion in cats. In vitro responses of middle cerebral arteries prepared from control animals and from animals subjected to hemorrhagic hypotension with or without specific PAF antagonist WEB 2086 treatment (1 mg/kg initial bolus followed by a 0.05 mg/kg/min infusion) were studied by measuring isometric force in organ chambers containing Krebs-Henseleit solution (37°C, gassed with 95% O 2– 5% CO 2). Bleeding was performed in a stepwise fashion by bringing the mean arterial blood pressure to 90, 70 and 50 mmHg and maintained for 20 min at each level followed by a 20-min retransfusion. Hemorrhagic hypotension and retransfusion caused a marked attenuation of the acetylcholine- and ATP-induced endothelium-dependent relaxations of the middle cerebral artery whereas the dilations induced by the nitric oxide donor and direct vasorelaxant SIN-1, remained unaltered. In the vessels, prepared from animals which received WEB 2086 treatment during hemorrhage and retransfusion there were more pronounced cholinergic (but not purinergic) relaxations than in the untreated animals subjected to hemorrhage. SIN-1 induced relaxations remained unaltered after WEB 2086 treatment. Our results suggest that platelet-activating factor is in part involved in the pathophysiological processes leading to the development of the endothelial dysfunction in the present model of hemorrhagic hypotension and retransfusion.

Anoxia-reoxygenation-induced, neutrophil-mediated endothelial cell injury: role of elastase

The aim of this study was to assess the role of neutrophilic elastase in anoxia-reoxygenation-induced, neutrophil-mediated injury to microvascular endothelium. Cultured bovine microvascular endothelial cells were grown to confluence and labeled with 51Cr. The endothelial cells were exposed to a 30-min period of anoxia and subsequently reoxygenated. Endothelial cell injury, quantitated as 51Cr release and cell detachment, was determined 8 h after reoxygenation. Addition of neutrophils upon reoxygenation enhanced the anoxia-reoxygenation-induced increase in 51Cr release and cell detachment. The neutrophil-mediated injury was associated with elastase release from the neutrophils. Four agents were used to inhibit neutrophilic elastase activity: Eglin C, methoxysuccunyl-Ala2-Pro-Val-CH2Cl, L658,758, and a monoclonal antibody against neutrophilic elastase. All elastase inhibitors attenuated the neutrophil-mediated endothelial cell detachment but not 51Cr release. Addition of purified human neutrophilic elastase, at a level that mimicked the release from neutrophils, increased cell detachment in endothelial cells exposed to anoxia-reoxygenation but did not affect 51Cr release. Our results indicate that elastase plays an important role in anoxia-reoxygenation-induced, neutrophil-mediated endothelial cell dysfunction.

Ethanol-induced injury to the rat gastric mucosa: Role of neutrophils and xanthine oxidase-derived radicals

Ethanol-induced gastric mucosal injury closely resembles an inflammatory response. Thus, in vivo and in vitro experimental models were used to assess whether ethanol is proinflammatory in concentrations likely to be encountered by the gastric mucosa during acute intoxication. Perfusing the rat gastric lumen with progressively increasing concentrations of ethanol (10%, 20%, and 30%) resulted in a dose-dependent increase in 51Cr-ethylenediaminetetraacetic acid clearance from blood-to-gastric lumen. Rendering the animals neutropenic (with antineutrophil serum) ameliorated the ethanol-induced mucosal injury; the degree of protection was directly related to the severity of neutropenia. Neither superoxide dismutase, catalase, nor sodium benzoate offered any protection against ethanol-induced injury, indicating that neither superoxide anion, hydrogen peroxide, nor the hydroxyl radical is involved. To assess further whether ethanol could exert proinflammatory effects an in vitro model consisting of cultured bovine microvascular endothelial cells and isolated human neutrophils was used. Ethanol at concentrations of 1.0%–4.0% (but not at 0.1%–0.5%) increased neutrophil adherence to endothelial cells and enhanced neutrophil-mediated endothelial cell injury. We conclude that ethanol is proinflammatory at concentrations that may be achieved in the gastric mucosa during acute intoxication. The ethanol-induced, neutrophil-mediated cell injury does not appear to involve oxy radicals.

The role of neutrophil membrane glycoprotein 150 (Gp-150) in neutrophil-mediated endothelial cell injury in vitro.

In this study we examined the importance of neutrophil adherence in neutrophil-mediated endothelial cell injury. Phorbol myristate acetate (PMA)-activated neutrophils from a patient with a congenital defect in neutrophil adherence (Gp-150 deficiency) and PMA-activated normal neutrophils pretreated with monoclonal antibody (MoAb) 60.3 were used. Both Gp-150-deficient and MoAb 60.3-treated normal neutrophils failed to adhere to cultured human umbilical vein endothelial cell (HEC) monolayers when activated by PMA (adherence less than 10% with patient and MoAb 60.3-treated cells compared with 53 +/- 3% with normal cells). The addition of PMA-activated normal neutrophils to 51Cr-labeled HEC monolayers failed to induce significant 51Cr release but did produce marked HEC detachment (percentage of detachment 50 +/- 3 at 6 hr). In marked contrast, PMA-activated Gp-150-deficient neutrophils failed to induce significant HEC detachment (percentage of detachment zero (0) at 6 hr). Moreover, the addition of MoAb 60.3 to normal neutrophils inhibited neutrophil-mediated HEC detachment in a time- and dose-dependent fashion. Non-lytic HEC detachment was determined to be largely oxygen radical independent, because PMA-activated chronic granulomatous disease neutrophils and PMA-activated normal neutrophils produced similar disruption of HEC monolayers. Soybean trypsin inhibitor, a chloromethylketone elastase inhibitor, and autologous serum all failed to inhibit neutrophil-mediated HEC detachment. From these studies there is no evidence that nonlytic HEC detachment by PMA-activated neutrophils is mediated by the neutrophil-derived proteases, elastase and cathepsin G. Neutrophil-mediated HEC detachment also required intact neutrophils, because postsecretory medium from PMA-activated normal neutrophils and a suspension of frozen-thawed PMA-activated normal neutrophils were without effect. These in vitro studies indicate that the neutrophil cell surface glycoprotein Gp-150 is required for nonlytic HEC detachment by intact PMA-activated neutrophils.

Neutrophils kill pulmonary endothelial cells by a hydrogen-peroxide-dependent pathway. An in vitro model of neutrophil-mediated lung injury.

Neutrophil-mediated injury to lung parenchymal cells has been proposed as an important step in the pathogenesis of many acute and chronic lung disorders. As an in vitro model of neutrophil-mediated injury, this study used activated human neutrophils as effector cells in an 18-h cytotoxicity assay with 51Cr-labeled bovine pulmonary artery endothelial cells serving as target cells. Neutrophils effectively injured pulmonary endothelial cells, expressed as cytotoxic index (CI), of 63.8 +/- 5.4, and this injury could be significantly reduced by several agents, including 1% dimethyl sulfoxide (CI, 51.3 +/- 3.7), 50 micrograms/ml ascorbic acid (CI, 40.8 +/- 4.7), and especially 1,100 U/ml catalase (CI, 14.3 +/- 4.1). As cell-free models of neutrophil-mediated endothelial cell injury, H2O2 (30 microM), O2- (generated by 0.5 mU xanthine oxidase), and the myeloperoxidase-dependent (0.32 U) hypohalite ion were each capable of injuring the target cells with CI of 6.21 +/- 2.8, 53.6 +/- 5.3, and 21.2 +/- 1.5, respectively. Catalase was effective in reducing the injurious effect of each of these oxidant-generating systems (p less than 0.01, all comparisons), confirming the important role for H2O2 in the mediation of this injury. The data indicate that neutrophils are capable of killing pulmonary endothelial cells by a pathway largely dependent on the generation of H2O2, and suggest the possibility that removal of H2O2 from the alveolar structures in subjects with these disorder might be an effective future therapeutic approach.