Source of iron in neutrophil-mediated killing of endothelial cells

Recently we have shown that human neutrophils activated with phorbol ester are cytotoxic for cultured bovine pulmonary artery endothelial cells in an iron-dependent manner. By using the ferric iron chelator deferoxamine mesylate, we have now investigated the source of the iron. Pretreatment of neutrophils with deferoxamine mesylate affected neither their production of O2- nor their cytotoxicity for endothelial cells after addition of phorbol ester. However, similar pretreatment of endothelial cells with deferoxamine mesylate, followed by washing of the cells, resulted in a persistent presence of chelator associated with the endothelial cells and high degrees of protection of endothelial cells from cytotoxicity. The protection was dependent on the amount of chelator used and on the duration of exposure of the endothelial cells to the chelator. These data suggest that iron, which plays an important role in oxygen radical-mediated killing of endothelial cells by neutrophils, is derived from the target (endothelial) cells.     

Evidence for a role of hydroxyl radical in immune-complex-induced vasculitis.

Previously it was shown that tissue injury occurring in acute immune-complex-induced vasculitis, which is complement and neutrophil-dependent, is significantly attenuated by the presence of catalase, suggesting the pathogenic role of H2O2 generated from activated neutrophils. We now show that significant protection is also afforded by pretreatment of animals with apolactoferrin , a naturally occurring chelator of iron. Iron-saturated lactoferrin is devoid of protective effects. Deferoxamine mesylate, a synthetic iron chelator, also has protective effects. Infusion of ionic iron, especially Fe(III), potentiates the tissue injury. Significant protection from tissue injury is also produced by treatment of rats with dimethyl sulfoxide, a potent hydroxyl radical scavenger. Morphologically, animals treated with these protective interventions show the influx of neutrophils into sites of immune complex deposition, but there is markedly attenuated edema, little or no hemorrhage, and little evidence of endothelial cell injury, in contrast to the findings in nonprotected animals. These data support the suggestion that immune-complex-induced injury may be linked to generation of H2O2 from activated neutrophils and the subsequent conversion of H2O2 to the hydroxyl radical.     

Human umbilical vein endothelial cell killing by activated neutrophils. Loss of sensitivity to injury is accompanied by decreased iron content during in vitro culture and is restored with exogenous iron.

First passage human umbilical vein endothelial cells (HUVECs) were sensitive to killing by activated neutrophils and reagent hydrogen peroxide (H2O2). Catalase and deferoxamine prevented killing whereas soybean trypsin inhibitor and superoxide dismutase did not. In these regards, HUVECs are similar to previously characterized endothelial cells from bovine and rat. Although first passage HUVECs were killed by activated neutrophils, sensitivity fell off rapidly as the cells were maintained in culture. At passage 2 (four population doublings), and beyond, HUVECs were highly resistant. The cells also became resistant to killing by reagent H2O2. The acquisition of resistance to killing was not accompanied by a failure to up-regulate neutrophil adhesion molecules or to support neutrophil adhesion. Levels of intracellular anti-oxidants (total thiols, though not glutathione, glutathione peroxidase or catalase activity) increased as a function of passage in culture. However, levels of glutathione and total thiols in late passage (resistant) HUVECs were similar to levels in late passage rat pulmonary artery endothelial cells, that were sensitive to killing by activated neutrophils. Cell-associated iron in HUVECs fell as a function of time in culture. By passage 2, the amount of total iron measurable with the Ferrozine reagent was only about 30% of the amount recovered from first passage HUVECs. The loss of iron from the cells may underlie much of the concomitant resistance to killing because when the cells were pretreated with iron under conditions in which it could be taken up, sensitivity to killing by activated neutrophils and by H2O2 was restored.     

Involvement of reactive oxygen metabolites in the candidacidal activity of human neutrophils stimulated by muramyl dipeptide or tumor necrosis factor

In the presence of the adjuvant glycopeptide muramyl dipeptide (MDP), purified human PMN exhibited an enhanced capacity to kill Candida albicans cells at various cell ratios. A significant effect was obtained at 100 ng/ml MDP, and the maximum was reached at 1 micrograms/ml MDP. Recombinant human tumor necrosis factor (rHuTNF), a monokine that enhances host resistance to bacterial and fungal infections, also stimulated the candidacidal potency of PMN with a maximal effect at 10(-2) ng/ml rHuTNF. When MDP- or rHuTNF-stimulated PMN were cultured with yeast cells, the intracellular production of oxygen metabolites was enhanced. Pretreatment with inhibitors of oxidative burst demonstrated that the yeast cell killing by MDP-stimulated PMN was not affected by SOD but was inhibited by sodium azide, indicating the involvement of myeloperoxidase (MPO)-halide system in fungicidal mechanisms induced by MDP. When PMN were stimulated with rHuTNF, the killing of yeast cells was neutralized by iodoacetamide, showing that the candidacidal potency of stimulated-PMN was due to oxygen derivatives. Inhibition by sodium azide and sodium benzoate indicated that these oxygen metabolites could be derived from the MPO-halide system but also from hydroxyl radical production. Moreover, SOD partially inhibited the fungicidal potency of rHuTNF-stimulated PMN, thus indicating a possible reutilization of the released O2- anion for intracellular killing. Cytochalasin B abrogated the PMN fungicidal potency in all cases.     

Resistance of rat pulmonary alveolar epithelial cells to neutrophil- and oxidant-induced injury

We have previously reported that rat pulmonary alveolar epithelial cells are resistant to neutrophil-generated oxidants in contrast to the situation described for endothelial cells. In the present study, we investigated the roles of intracellular catalase and glutathione-dependent reactions in providing protection against cytotoxic concentrations of H2O2 and stimulated neutrophils. Catalase was found to be instrumental in protecting epithelial cells because when inhibited by either azide or 3-amino-1,2,4-triazole, there was an increase in the cytotoxic effect of exogenous H2O2 and stimulated neutrophils. Associated with this potentiation of injury was a reduction in epithelial cell clearance of H2O2. Partial inhibition of glutathione-dependent reactions by depleting intracellular glutathione with buthionine sulfoximine or by inhibiting the enzyme glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea also augmented the cytotoxic effect of both H2O2 and stimulated neutrophils. This increase in neutrophil-induced cytotoxicity was caused by the addition of an oxidant-dependent mechanism of killing on top of the previously described oxidant-independent pathway. Importantly, the increased susceptibility to injury caused by inhibition of glutathione-dependent reactions was not associated with a reduction in epithelial cell consumption of exogenous H2O2, contrary to the case with catalase. This suggests that there are glutathione-dependent reactions that protect epithelial cells in ways separate from reducing the total burden of exogenous H2O2 on the cells.     

Xanthine oxidase activity in rat pulmonary artery endothelial cells and its alteration by activated neutrophils.

The possibility that endothelial cell-derived oxidants could contribute to neutrophil-mediated endothelial cell injury and cytotoxicity has been a subject of speculation. Rat pulmonary artery endothelial cells (RPAECs) were examined for the presence of xanthine oxidase (XO) activity, a well-known source of O2-. Using a sensitive assay based on measurements of radioactive xanthine conversion to uric acid by high performance liquid chromatography (HPLC), RPAEC extracts were found to contain both XO and xanthine dehydrogenase (XD) activities. Extracts from early passage cells have 55.3 +/- 11.7 (mean +/- SE) units/10(6) cells of total (XO + XD) activity, one unit of activity being defined as the conversion of 1% of substrate to product in 30 minutes of incubation. XO comprised 31.6 +/- 3.1% of this total activity. Addition of human neutrophils stimulated with phorbol myristate acetate (PMA) caused a rapid and dose-dependent increase in RPAEC XO activity from 31.6 +/- 3.1% to 71.7 +/- 4.8% of total without altering total (XO + XD) activity. The neutrophil dose-response curve for increase in XO paralleled closely the curve for neutrophil-mediated RPAEC cytotoxicity. The basal XO and XD activities and the neutrophil-induced increase in XO activity were inhibited by treating RPAECs with allopurinol, oxypurinol, and lodoxamide, which also inhibited cytotoxicity, but not by catalase, superoxide dismutase, or deferoxamine. Addition of H2O2 failed to cause an increase in RPAEC XO activity or XD to XO conversion. The results suggest that during neutrophil-mediated injury, rapid conversion of RPAEC XD to XO occurs, resulting in increased XO, catalyzed endogenous oxidant production, which may contribute to the oxidant burden in the killing mechanism initiated by activated neutrophils. Although the mechanism for conversion of XD to XO is uncertain, it appears that neutrophil-derived H2O2 is not sufficient to cause this phenomenon. Furthermore, neither O2- nor chelatable iron is required for neutrophil-induced XD to XO conversion. Supernatant fluids from activated neutrophils failed to induce XD to XO conversion in RPAECs. This in vitro system provides an opportunity to define the cellular and molecular mechanisms underlying the in vivo phenomenon of XD to XO conversion associated with ischemic/reperfusion or inflammatory tissue injury.     

Effect of environmental particulates on cultured human and bovine endothelium. Cellular injury via an oxidant-dependent pathway

The effects of respirable environmental fibers on cultures of human umbilical vein and bovine pulmonary artery endothelial cell monolayers were studied. Interaction among endothelial cell monolayers and amosite and chrysotile asbestos, attapulgite, fiberglass, or latex beads resulted in rapid phagocytosis of the particulates. A gradient of time-dependent and concentration-dependent endothelial cell injury (measured by specific 51Cr release) was observed with amosite and attapulgite being markedly toxic. Chrysotile and fiberglass were much less toxic, and latex beads were not significantly injurious at any time or dose examined. Responses of bovine pulmonary artery and human endothelial vein endothelial cells to fiber phagocytosis and fiber-induced injury were similar. In human umbilical cell monolayers, fiber-mediated stimulation of the arachidonate metabolite prostacyclin paralleled endothelial cell injury; i.e. amosite and attapulgite were stimulatory, whereas fiberglass (0-500 micrograms/ml) and latex beads (10(9) beads/ml) did not significantly increase prostacyclin generation. Although chrysotile was only weakly cytotoxic, significant stimulation of prostacyclin was observed at the highest dose tested (500 micrograms/ml). To investigate whether toxic oxygen species may be involved in fiber-induced cytotoxicity, oxidant scavengers or inhibitors were used in injury studies. Both superoxide dismutase (a scavenger of O2-) and catalase (an inhibitor of H2O2) produced significant protection against fiber-mediated endothelial cell injury. In addition, chelation by deferoxamine of elemental Fe present in the fiber preparations was also protective, suggesting Fe, via the modified Haber-Weiss reaction, may promote hydroxyl radical formation and contribute to endothelial cell injury induced by these particulates. These results suggest that the interaction within the interstitial environment between endothelial cells and occupationally relevant dusts may be important in fiber-mediated inflammatory processes in the lung.     

Cytotoxicity of tumor necrosis factor-alpha for human umbilical vein endothelial cells

Human umbilical vein endothelial cells were examined for sensitivity to killing by human recombinant tumor necrosis factor-alpha (TNF-alpha). Treatment of the cells with concentrations of TNF-alpha up to 50 ng/ml for 18 hours did not produce evidence of cytotoxicity. However, a marked cytotoxic effect was found when TNF-alpha pretreated cells were incubated in Hanks' balanced salt solution for a further 4 hours. Exposure of the cells to heat-inactivated or antibody-neutralized TNF-alpha did not result in cytotoxicity. Human recombinant interleukin-1 also lysed endothelial cells under the same conditions, whereas human recombinant macrophage-colony stimulating factor did not. Inclusion of superoxide dismutase, catalase, or soybean trypsin inhibitor in the culture medium during the time of endothelial cell exposure to TNF-alpha had no protective effects. Likewise, allopurinol (a xanthine oxidase inhibitor) and nordihydro-guaiaretic acid (a lipoxygenase inhibitor) were not protective under the same conditions. In contrast, the ferric iron chelator deferoxamine mesylate and three different cyclooxygenase inhibitors provided significant protection against TNF-alpha induced cytotoxicity. When human dermal fibroblasts and human squamous epithelial cells were used in place of the umbilical vein endothelial cells, these cells were resistant to TNF-alpha mediated killing. These findings demonstrate that under the experimental conditions employed, TNF-alpha is cytotoxic for human umbilical vein endothelial cells. This may have implications in a number of in vivo situations in which TNF-alpha is thought to play a role.     

Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin.

Pseudomonas aeruginosa causes acute and chronic infections of the human lung, with resultant tissue injury. We have previously shown that iron bound to pyochelin, a siderophore secreted by the organism to acquire iron, is an efficient catalyst for hydroxyl radical (HO.) formation and augments injury to pulmonary artery endothelial cells resulting from their exposure to superoxide (O2.) and/or H2O2. Sources for O2-. and H2O2 included phorbol myristate acetate (PMA)-stimulated neutrophils and pyocyanin. Pyocyanin, another P. aeruginosa secretory product, undergoes cell-mediated redox, thereby forming O2-. and H2O2. In P. aeruginosa lung infections, damage to airway epithelial cells is probably more extensive than that to endothelial cells. Therefore, we examined whether ferripyochelin also augments oxidant-mediated damage to airway epithelial cells. A549 cells, a human type II alveolar epithelial cell line, was exposed to H2O2, PMA-stimulated neutrophils, or pyocyanin, and injury was determined by release of 51Cr from prelabeled cells. Ferripyochelin significantly increased (> 10-fold) oxidant-mediated cell injury regardless of whether H2O2, neutrophils, or pyocyanin was employed. Apo-pyochelin was not effective, and ferripyochelin was not toxic by itself at the concentrations employed. Spin trapping with alpha-(4-pyrridyl-1-oxide)-N-t-butyl-nitrone-ethanol confirmed the generation of HO., and injury was decreased by a variety of antioxidants, including superoxide dismutase, catalase, and dimethylthiourea. These data are consistent with the hypothesis that the presence of ferripyochelin at sites of P. aeruginosa lung infection could contribute to tissue injury through its ability to promote HO.-mediated damage to airway epithelial cells.     

Specific binding of haptoglobin to human neutrophils and its functional consequences

Haptoglobin, an acute phase reactant protein, has been shown to modulate various facets of immune responses. In this paper we examined the effect of haptoglobin on human neutrophils at the molecular level. First, we found that native haptoglobin binds at two distinct sites on neutrophils. We then examined the effects of this binding at normal and pathophysiological concentrations of haptoglobin found in human serum. Of the various functional parameters assessed, neutrophil respiratory burst activity, as assessed by superoxide (O2-) production, was inhibited by native haptoglobin when the cells were stimulated with formylmethionyl-leucylphenylalanine (FMLP), arachidonic acid (AA), and opsonized zymosan. The rise in intracellular calcium induced by FMLP stimulation was also inhibited by native haptoglobin. Since the generation of O2- was unaffected by native haptoglobin in phorbol myristate acetate (PMA)-stimulated neutrophils, the likely site of haptoglobin inhibition on neutrophil function is at a point of receptor-ligand interaction in the activation cascade. The role of haptoglobin as a modifier of the immune response has here been extended to altered neutrophil function stimulated by diverse agonists.     

Protection by deferoxamine from endothelial injury: a possible link with inhibition of intracellular xanthine oxidase

Hydroxyl radical scavengers and xanthine oxidase inhibitors protect cultured bovine pulmonary endothelial cells (BPAEC) from lytic injury by the endotoxin lipopolysaccharide (LPS). We hypothesized that exposure of BPAEC to cytotoxic concentrations of LPS activated intracellular xanthine oxidase, and that intracellular iron-dependent hydroxyl radical formation (a Fenton reaction) ensued, resulting in cell lysis. To test this, the protective effects of deferoxamine against H2O2 and LPS-induced cytotoxicity to BPAEC was assessed by 51Cr release. Preincubation with 0.4 mM deferoxamine conferred 67 +/- 15% (mean +/- SE) protection from LPS-induced cytotoxicity but 48 h of preincubation were required to induce significant protection. Significant protection form a classical Fenton reaction model, injury by 50 microM H2O2, could be induced by a 1-h preincubation with a 0.4 mM deferoxamine. The dissociated time course suggested that deferoxamine might work by different mechanisms in these models. The effects of LPS and deferoxamine on BPAEC-associated xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity were assessed using a spectrofluorophotometric measurement of the conversion of pterin to isoxanthopterin. BPAEC had 106 +/- 7 microU/mg XD+XO activity; XO activity constituted 48 +/- 1% of total XO+XD activity. LPS at a cytotoxic concentration did not alter XO, XD, or percent XO. Deferoxamine had striking proportional inhibitory effects on XO and XD in intact cells. XO+XD activity fell to 6 +/- 1% of control levels during a 48-h exposure of BPAEC to deferoxamine. Deferoxamine did not inhibit XO+XD ex vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor

Confluent monolayers of bovine pulmonary artery endothelial cells (BPAE) or human umbilical vein endothelial cells (HUVE) inhibited by 80 to 90% the production of O2- by added human neutrophils (PMNs) stimulated by plasma membrane receptor-mediated activators (formylmethionylleucylphenylalanine [fMLP], opsonized zymosan, heat-killed Staphylococci), but not by non-plasma membrane receptor-mediated activators (phorbol myristate acetate and delta-hexachlorocyclohexane). Degranulation induced by fMLP was also inhibited by BPAE. Inhibition was not affected by eicosatetraynoic acid (ETYA) or indomethacin. To assess the role of cell-cell contact, 0.45-microns-pore culture plate inserts were employed to prevent PMN-endothelial cell contact during incubation. A similar amount of inhibition of stimulated PMNs superoxide production was seen as compared to PMN-endothelial incubations where contact occurred. A soluble component released by BPAE monolayers, when added to PMNs, duplicated the inhibition seen by BPAE-PMN co-incubation. Incubation of BPAE with adenosine deaminase did not reduce inhibition of O2- production compared to controls without adenosine deaminase. There was no evidence of endothelial scavenging of O2- generated by hypoxanthine-xanthine oxidase, and inhibition of endothelial superoxide dismutase did not diminish the inhibitory effort. We conclude that cell contact is not required for BPAE inhibition of fMLP-stimulated O2- production by PMN, and that scavenging of superoxide anion is not the mechanism. The inhibitor appears to be a polypeptide with an apparent molecular weight between 1,000 and 10,000 D and does not appear to be adenosine, an arachidonate metabolite, or superoxide dismutase. The mechanism may involve down-regulation of plasma membrane receptor-mediated activation of PMNs.     

Protease inhibitors (gebexate mesylate and ulinastatin) stimulate intracellular chemiluminescence in human neutrophils.

The effect of protease inhibitors on the intracellular production of free radicals was investigated by measuring chemiluminescence (CL) elicited from phagocytosed luminol-bound microspheres (Lumispheres) in human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), phorbol 12-myristate 13-acetate, or diacylglycerol. Both gabexate mesylate (Foy) and ulinastatin (Miraclid), urinary trypsin inhibitor, increased intracellular CL in a dose dependent manner. Compared to control buffer without protease inhibitor, gabexate mesylate (322 micrograms/ml) caused about a 10-fold increase in intracellular CL in stimulated neutrophils, and ulinastatin (3100 U/ml) a twofold increase in neutrophils stimulated with fMLP or IL-8. When the protease inhibitors were added to the cell suspension after the phagocytosis of lumispheres, CL responses rapidly increased again to the level which was observed when both protease inhibitors and neutrophil stimulants were incubated simultaneously. In contrast, extracellular release of oxygen metabolites from stimulated neutrophils, assayed by a conventional measurement of luminol-dependent CL, was reduced by the protease inhibitors in a dose dependent fashion. When luminol-unbound microspheres were incubated with neutrophils stimulated by fMLP in luminol solution, extracellular CL was almost completely inhibited by gabexate mesylate. These results indicate that the protease inhibitors enhance the generation of intracellular CL and suppress the extracellular release of free radicals.     

Lipid peroxidation and acute lung injury after thermal trauma to skin. Evidence of a role for hydroxyl radical.

The authors have previously shown that thermal injury to the skin of rats results in the development of acute lung injury that is susceptible to systemic treatment of animals with catalase and dependent on the presence of neutrophils. The current studies have been expanded for exploration of the nature of the neutrophil-derived oxygen products responsible for the lung injury and have also focused on evidence of the appearance of products of lipid peroxidation (conjugated dienes). With respect to the former, treatment of rats with iron chelators (deferoxamine mesylate, 2,3-dihydroxybenzoic acid), with scavengers of hydroxyl radical (dimethyl sulfoxide, dimethyl thiourea, sodium benzoate), or with vitamin E affords a significant degree of protection from acute lung injury as assessed by changes in lung vascular permeability and by morphologic parameters. These data suggest that lung vascular injury after thermal trauma of the skin is related to the generation by neutrophils of the hydroxyl radical. Conjugated dienes have been demonstrated to appear sequentially both in the burned skin (at 1/4 hour) and in the lungs (at 2 hours), as well as in the plasma (with peaks at 1/2 and at 3 hours) after thermal injury. The appearance of the conjugated dienes in plasma at the two intervals of time is greatly diminished if animals are pretreated with the iron chelator deferoxamine, with catalase, or with scavengers of hydroxyl radical. Furthermore, the appearance of conjugated dienes in plasma at 30 minutes and 3 hours is significantly diminished if animals are depleted of neutrophils, complement-depleted, or the burned skin is excised immediately after thermal injury. These data indicate a linkage between thermal trauma of skin, secondary injury of lung, and appearance in plasma and tissues of products of lipid peroxidation.     

Effect of biological surfaces on neutrophil O2- production and its relationship to the CD11b/CD18 integrin-dependent adherence.

The production of O2- in response to LPS, PAF, FMLP, TNF and PMA by human neutrophils in suspension and residing on surfaces coated with fetal calf serum (FCS), fibronectin (FN), laminin (LM), collagen types I and IV (CI and CIV), fibrinogen (FBG) or fibrin (FBN) was studied. Of the agonists used, PAF and LPS failed to induce a response in any of the above conditions; FMLP and PMA stimulated neutrophils to produce similar amounts of O2- either in suspension or on biological surfaces; TNF induced O2- production only by cells residing on FN, FBG and FBN. These results indicate that production of oxygen-derived free radicals by neutrophils depends on the type of agonist and the nature of the surface they interact with. The relationship between the respiratory burst and adherence was studied by measuring O2- release and adherence of neutrophils residing on FN, LM, CIV, and FBG, in the absence and in the presence of the monoclonal antibody 60.3 that recognizes the common beta-chain of CD11/CD18 integrins. FMLP, PMA and TNF increased neutrophil adherence on all these surfaces except CIV. The monoclonal antibody markedly inhibited the FMLP and PMA-induced adherence but had no effect on the O2- release elicited by these two agonists. In contrast, the monoclonal antibody inhibited both the increased adherence and O2- release induced by TNF on FN and FBG. The TNF-induced increase in adherence to LM, that was not accompanied by an increase in O2- release, was also inhibited by the monoclonal antibody. We conclude that the respiratory burst of neutrophils residing on surfaces is not necessarily correlated with adherence.     

Enhancement of neutrophil-mediated injury to bovine pulmonary endothelial cells by Pasteurella haemolytica leukotoxin.

In this study, we used an in vitro coculture system to determine which virulence factor from Pasteurella haemolytica A1 was responsible for augmenting bovine polymorphonuclear neutrophil (PMN)-mediated killing of bovine pulmonary artery endothelial cells (BPAEC). A 51Cr release cytotoxicity assay was used as a measure of BPAEC killing. The mechanisms associated with this BPAEC killing were also studied. Our results demonstrated that the leukotoxin and not the lipopolysaccharide from P. haemolytica was responsible for augmenting the PMN-mediated killing of BPAEC. Furthermore, this augmented killing was related to the stimulation of PMNs by the leukotoxin. Killing of BPAEC by leukotoxin-stimulated PMNs was diminished in the presence of the H2O2 inactivator, catalase. The membrane-permeant H2O2, hydroxyl radical (HO.) scavenger 1,3-dimethyl-2 thiourea, and the HO. scavenger dimethyl sulfoxide but not the myeloperoxidase inhibitor sodium azide attenuated this BPAEC killing. Pretreatment of BPAEC with a 21-aminosteroid (U74500A), a potent iron chelator-antioxidant, provided the most effective protection against BPAEC killing induced by leukotoxin-stimulated PMNs. These data were compatible with the concept that the H2O2 generated by leukotoxin-stimulated PMNs interacts with intracellular iron in the endothelial cell to form highly reactive HO.. We suggest that HO. may be a key factor in BPAEC killing. Furthermore, since the elastase-specific inhibitor N-methoxy-succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (CMK) also attenuated BPAEC killing and both CMK and 1,3-dimethyl-2 thiourea functioned additively in protecting against BPAEC killing, we conclude that both HO. and elastase may jointly contribute to BPAEC killing induced by leukotoxin-stimulated PMNs. This study broadens our understanding of how leukotoxin-stimulated PMNs injure lung endothelial cells and provides new insight into the pathogenesis of bovine pneumonic pasteurellosis.     

Activated rat neutrophils. Correlation of arachidonate products with enzyme secretion but not with O(2)- generation.

Functional responses (enzyme secretion and generation of O2-) have been studied in rat neutrophils with the use of a variety of different agonists which vary in their ability to activate neutrophils. Concomitantly, the authors have analyzed the cells for production of cyclooxygenase (PGE2, PGF2 alpha, and TXB2) and lipoxygenase (monoHETEs and LTB4) products in order to determine whether there is a correlation between functional responses and arachidonate products in stimulated neutrophils. The studies indicate that enzyme secretion is closely correlated with generation of arachidonate (cyclooxygenase as well as lipoxygenase) products. These responses are dependent on the dose of agonist employed, but there does not appear to be a unique pattern of arachidonate products that can be attributed to a specific agonist. With respect to monohydroxyeicosatetraenoic acid (monoHETE) generation, we demonstrate that stimulated rat neutrophils selectively produce 5-HETE, to the virtual exclusion of 12-HETE and 15-HETE. The production of O2- from stimulated rat neutrophils is correlated neither with enzyme secretion nor with the generation of arachidonate products. These studies suggest that enzyme secretion and O2- generation are mediated by two fundamentally different intracellular pathways, even though these functional responses probably derive from common agonist-receptor interactions on the plasma membrane.     

Mediation of IgA induced lung injury in the rat. Role of macrophages and reactive oxygen products

Acute lung injury in the rat has been induced by the instillation of affinity-purified mouse monoclonal IgA antibody with specific reactivity to the hapten dinitrophenol coupled to albumin. As previously reported, this model of lung injury requires an intact complement system but is independent of neutrophils. In contrast to macrophages obtained by bronchoalveolar lavage from rats receiving IgA into the airways in the absence of intravenously injected antigen, macrophages obtained from the lungs of rats developing IgA immune complex-induced lung injury were significantly increased in number, showed greater spontaneous generation of O2.-, and demonstrated significantly enhanced O2.- responses in the presence of an added stimulus, phorbol ester. Inhibition studies in vivo suggested that the IgA-induced lung injury is mediated by oxygen radicals generated from lung macrophages. Pretreatment of animals with superoxide dismutase, catalase, the iron chelator, deferoxamine, or the hydroxyl radical scavenger, dimethyl sulfoxide, suppressed the development of lung injury. Morphologically the lungs of protected animals showed increased numbers of mononuclear cells within the alveolar compartment but little evidence of alveolar or capillary injury, in contrast to unprotected animals in which there was evidence of severe injury, both to microvascular interstitial endothelial cells as well as to alveolar lining epithelial cells. These studies suggest that acute lung injury in the rat induced by IgA immune complexes is mediated by oxygen radical formation and that the macrophage may be the principle effector cell, as compared to IgG immune complex induced lung injury, which is also oxygen radical mediated but in which the neutrophil is the effector cell.     

Adiponectin inhibits superoxide generation by human neutrophils

Adiponectin (Ad), a member of the adipocytokine family, has been reported to possess antiinflammatory properties. We investigated the effects of full-length human Ad (hAd) on phorbol 12-myristate 13-acetate (PMA)-induced O2-* generation by human neutrophils. hAd, even at the lowest tested concentration of 0.001 microg/ml, after 30-min pretreatment of cells, significantly inhibited O2-* generation by neutrophils stimulated with PMA (100 nM). However, no relation between the dose of hAd and extent of inhibition of PMA-induced O2-* generation was observed with increasing the concentration of hAd up to 1 microg/ml. hAd also significantly inhibited neutrophil O2-* generation stimulated by N-formyl-methionyl-leucyl-phenylalanine (100 microM) and diacylglycerol (500 nM), as well as the PMA-induced neutrophil nitroblue tetrazolium reduction and H2O2 formation. Pretreatment of neutrophils with pronase-digested hAd failed to inhibit the PMA-induced O2-* generation. For the first time, this study revealed that Ad inhibited O2-* generation by neutrophils, possibly through regulation of NADPH oxidase.     

Inhibitory effects of vasoactive intestinal peptide on superoxide anion generation from stimulated human inflammatory cells]

Vasoactive intestinal peptide (VIP) is suspected to be a neurotransmitter of nonadrenergic and noncholinergic inhibitory nerves in human respiratory tracts. Although VIP affects T lymphocytes through its specific receptor, the effects of VIP on inflammatory cells except T lymphocytes are obscure. We investigated the effects of VIP on superoxide anion (O2-) generation from five kinds of human cells; neutrophils, eosinophils and mononuclear cells isolated from peripheral blood, alveolar macrophages obtained from the bronchoalveolar lavage, and human monocyte cell line, U937, the capacity of which for O2- generation was induced by interferon-gamma. O2- generation from human cells stimulated by 10(-5) M fMLP was measured by the cypridina luciferin analog, MCLA, -dependent chemiluminescence method. VIP inhibited O2- generation from fMLP-stimulated neutrophils, mononuclear cells and U937 in a dose-dependent manner. 3 x 10(-6) M VIP inhibited O2- generation from fMLP-stimulated eosinophils and alveolar macrophages significantly. These results indicate that VIP might inhibit the activation of inflammatory cells and act as an antiinflammatory agent.