p66(Shc) restrains Ras hyperactivation and suppresses metastatic behavior.

Normal tissue cells survive and proliferate only while anchored to solid substrate. Conversely, transformed cells both survive and proliferate following detachment, having lost attachment context through unclear mechanisms. p66(Shc) is a focal adhesion-associated protein that reports cell attachment through a RhoA-dependent mechanosensory test. We find that human small cell lung cancer (SCLC) cells and mouse Lewis lung carcinoma (LLC), which display aggressive metastatic behavior, lack both p66(Shc) and retinoblastoma (pRB) and bypass anoikis. Re-expression of p66(Shc) in these cells restores anoikis and provides striking protection from metastasis by LLC cells in vivo. Notably, knockdown of p66(Shc) in normal epithelial cells leads to unrestrained Ras activation, preventing anoikis through downstream suppression of RhoA but blocking proliferation in a pRB-dependent manner, thus mimicking oncogenic Ras. Conversely, LLC and SCLC cells display constitutive Ras activation necessary to bypass anoikis, which is reversed by re-expression of p66(Shc). p66(Shc) therefore coordinates Ras-dependent control of proliferation and anchorage sensation, which can be defeated in the evolution of highly metastatic tumors by combined loss of both p66(Shc) and pRB.

HIV Tat activates c-Jun amino-terminal kinase through an oxidant-dependent mechanism.

The HIV-1 accessory protein Tat has been found to exert profound effects on vascular cell behavior. Recently, Tat has been found to activate the c-Jun amino-terminal kinase (JNK1, SAPK) MAP kinase in lymphoid cells. We found that purified Tat rapidly activated JNK1 in human umbilical vein endothelial cells and ECV-304 cells, and coculture of ECV-304 cells with Tat-transfected HeLa cells resulted in persistent activation of JNK1. In addition, lower doses of Tat potentiated TNFalpha-induced JNK1 activation, although higher doses paradoxically diminished JNK1 activation by TNFalpha. Treatment of ECV-304 cells with Tat acutely increased intracellular oxidant levels, and Tat-induced oxidant activity was decreased by two structurally distinct NADPH oxidase inhibitors, diphenylene iodonium and apocynin. Both oxidase inhibitors and the thiol antioxidant N-acetyl cysteine decreased Tat-induced JNK1 activation in parallel with reduction in oxidant levels. Activation of JNK1 by Tat was also inhibited by cytochalasin B, suggesting that Tat signaling was dependent upon intact cytoskeletal function. Indeed, JNK1 activation by Tat was associated with actin microfilament rearrangement. We conclude that HIV Tat may cause acute and persistent activation of the JNK MAP kinase through activation of a specific oxidase.

Mechanism of free radical production in exhaustive exercise in humans and rats; role of xanthine oxidase and protection by allopurinol.

Exhaustive exercise generates free radicals. However, the source of this oxidative damage remains controversial. The aim of this paper was to study further the mechanism of exercise-induced production of free radicals. Testing the hypothesis that xanthine oxidase contributes to the production of free radicals during exercise, we found not only that exercise caused an increase in blood xanthine oxidase activity in rats but also that inhibiting xanthine oxidase with allopurinol prevented exercise-induced oxidation of glutathione in both rats and in humans. Furthermore, inhibiting xanthine oxidase prevented the increases in the plasma activity of cytosolic enzymes (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase) seen after exhaustive exercise. Our results provide evidence that xanthine oxidase is responsible for the free radical production and tissue damage during exhaustive exercise. These findings also suggest that mitochondria play a minor role as a source of free radicals during exhaustive physical exercise.

Platelet-activating factor contributes to acute lung leak in rats given interleukin-1 intratracheally.

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.

AIDS vasculopathy.

Persons infected with HIV display a variety of vascular abnormalities and harbor particularly striking alterations in endothelial morphology and function. We review the effects of the virus and viral products on the endothelium and emphasize their effects on altering the clinical expression of HIV-associated diseases.

Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity.

Pseudomonas aeruginosa is a persistent pathogen in the airways of patients with cystic fibrosis or bronchiectasis from other causes and appears to have evolved strategies to survive the inflammatory response of the host. We hypothesized that the secreted hemolytic phospholipase C (PLC) of P. aeruginosa (PlcHR) would decrease neutrophil respiratory burst activity. We found that while intact wild-type P. aeruginosa cells stimulated moderate respiratory burst activity from human neutrophils, an isogenic mutant pseudomonas (DeltaHR strain) containing a targeted deletion of the plcHR operon induced a much more robust oxidative burst from neutrophils. In contrast, a second pseudomonas mutant (DeltaN) containing a disruption in the gene encoding the nonhemolytic PLC (PlcN) was not different from the wild type in stimulating neutrophil O2.- production. Readdition of purified PlcHR to the DeltaHR strain suppressed neutrophil O2.- production to levels stimulated by wild-type bacteria. Interestingly, purified PlcHR decreased phorbol myristate acetate (PMA)- but not formyl methionyl-leucyl-proline (fMLP)-induced respiratory burst activity, suggesting interference by PlcHR with a protein kinase C (PKC)-specific signaling pathway. Accordingly, the PKC inhibitor bisindolylmaleimide inhibited the oxidative burst induced by either PMA or intact pseudomonas, but not by fMLP, whereas the p38 kinase inhibitor SB-203580 fully inhibited the respiratory burst induced by fMLP or the PlcHR-replete wild-type bacteria, but not PMA or the PlcHR-deficient DeltaHR bacterial mutant. We conclude that expression of PlcHR by P. aeruginosa suppresses bacterium-induced neutrophil respiratory burst by interfering with a PKC-dependent, non-p38 kinase-dependent pathway.

Diffuse alveolar hemorrhage with underlying pulmonary capillaritis in the retinoic acid syndrome.

All-trans-retinoic acid (ATRA) can induce a clinical remission in patients with acute promyelocytic leukemia. An adverse condition called "retinoic acid syndrome" limits this therapy. It is characterized by fever and respiratory distress, along with weight gain, pleural or pericardial effusions, peripheral edema, thromboembolic events, and intermittent hypotension. The lung disease has been previously ascribed to an infiltration of leukemic or maturing myeloid cells into lung parenchyma, which is sometimes associated with pleural effusions and diffuse alveolar hemorrhage. We report a case of retinoic acid syndrome in an 18-yr-old woman who developed diffuse alveolar hemorrhage while being treated with ATRA for acute promyelocytic leukemia. An open lung biopsy revealed pulmonary capillaritis.

Xanthine oxidase decreases production of gut wall nitric oxide.

Multiorgan failure is often the lethal outcome of intratracheal aspiration of acidic gastric juice. The pathogenesis of multiorgan failure may involve a systemic imbalance between pro-inflammatory and anti-inflammatory factors. In an anesthetized rat model, intratracheal instillation of HCl elicited intestinal inflammation which was exaggerated by xanthine oxidase (XO) and attenuated by nitric oxide (NO). We hypothesized that XO may mediate injury in part by suppression of NO formation. Therefore, we measured intestinal tissue concentrations of the stable NO oxidative metabolites (NO2- and NO3-) following intratracheal (IT) instillation of NaCl or HCl alone or in combination with interventions aimed at increasing or decreasing XO activity. Compared with IT NaCl (control treatment) jejunal tissue NO2- and NO2- + NO3- concentrations were increased by allopurinol pretreatment, which inhibits XO, and were decreased by systemically administered XO, as well as by IT HCl. The decreased NO2- and NO2- + NO3- concentrations found following IT HCl were completely reversed by either allopurinol or by systemically administered L-arginine (the precursor of NO). We conclude that manipulation of the pro-inflammatory XO system has a reciprocal effect on the intestinal anti-inflammatory NO system in either the undamaged or the endobronchially acidified lung model.

Hypoxia regulates xanthine dehydrogenase activity at pre- and posttranslational levels.

Hypoxia increases the activity of xanthine oxidase (XO) and its precursor, xanthine dehydrogenase (XDH), but the mechanism of regulation is unclear. In hypoxic Swiss 3T3 cells, an early (0-24 h) cycloheximide-insensitive increase in XO-XDH activity, coupled with a lack of increase in de novo XO-XDH synthesis (immunoprecipitation) or mRNA levels (quantitative RT-PCR), demonstrated a posttranslational effect of hypoxia. Similarly, hyperoxia decreased XO-XDH activity faster than could be accounted for by cessation of XO-XDH protein synthesis. In further support of a posttranslational effect, cells transfected with a constitutively driven XDH construct displayed an exaggerated increase in activity in hypoxia but no increase in activity in hyperoxia. However, more prolonged exposure to hypoxia (24-48 h) induced an increase in XO-XDH mRNA levels and de novo XO-XDH protein synthesis, suggesting an additional pretranslational effect. Finally, hypoxic induction of XO-XDH activity was found to be cell-type-restricted. We conclude that control of XO-XDH levels by oxygen tension is a complex process which involves several points of regulation.

Mepacrine decreases lung leak in rats given interleukin-1 intratracheally.

We hypothesized that phospholipase A2 (PLA2) metabolites contribute to the acute, neutrophil-dependent, edematous lung leak that develops after administration of interleukin-1 (IL-1) intratracheally to rats and tested this premise by using mepacrine to inhibit PLA2 activity in vivo. We found that lung PLA2 activity, lung lavage phospholipid content, lung leak index, lung weight gain, and lung lavage protein concentrations were increased in rats given IL-1 intratracheally compared with sham-treated control rats. By comparison, lungs of mecaprine and IL-1-treated rats had decreased PLA2 activity, lavage phospholipid content, leak, weight gain, and lavage protein increases compared with rats given IL-1 intratracheally. Mepacrine treatment also decreased lung neutrophil accumulation, but not lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, in rats given IL-1 intratracheally. In parallel experiments, mepacrine treatment reduced the adhesion of human neutrophils to IL-1-treated human umbilical vein endothelial cells in vitro. Our results indicate that PLA2 activity participates in the lung neutrophil retention and pulmonary vascular leak that develops in rats given IL-1 intratracheally.

Nitric oxide attenuates and xanthine oxidase exaggerates lung damage-induced gut injury.

Aspirated gastric contents can evoke multiorgan failure. We hypothesized that secondary intestinal epithelial dysfunction after lung damage would be mediated by xanthine oxidase (XO) and antagonized by endogenous gut nitric oxide (NO). Isosmotic saline or HCl solutions were instilled intratracheally in anesthetized rats, and intestinal injury was assessed 190 min later by measuring the blood-to-lumen clearance of 51Cr-labeled EDTA (51Cr-EDTA clearance) and gut wall neutrophil population density. Intratracheal HCl increased 51Cr-EDTA clearance, and this transepithelial leak was attenuated by either systemic L-arginine or intraluminal NO and by chronic dietary pretreatment with allopurinol or sodium tungstate. Conversely, lung damage-induced gut leak was exaggerated by NO synthase inhibition or intravenous XO administration. Intratracheal HCl also increased intestinal wall neutrophil density and myeloperoxide activity. We conclude that two enzymatic systems involved in remote gut barrier dysfunction after endobronchial acidification are XO as mediator and NO synthase as antagonist.

XO increases neutrophil adherence to endothelial cells by a dual ICAM-1 and P-selectin-mediated mechanism.

Circulating xanthine oxidase (XO) can modify adhesive interactions between neutrophils and the vascular endothelium, although the mechanism underlying this effect are not clear. We found that treatment with XO of bovine pulmonary artery endothelial cells (EC), but not neutrophils or plasma, increased adherence, suggesting that XO had its primary effect on EC. The mechanism by which XO increased neutrophil adherence to EC involved binding of XO to EC and production of H2O2. XO also increased platelet-activating factor production by EC by a H2O2-dependent mechanism. Similarly, the platelet-activating factor-receptor antagonist WEB-2086 completely blocked XO-mediated neutrophil EC adherence. In addition, neutrophil adherence was dependent on the beta 2-integrin Mac-1 (CD11b/CD18) but not on leukocyte functional antigen-1 (CD11a/CD18). Treatment of EC with XO for 30 min did not alter intercellular adhesion molecule-1 surface expression but increased expression of P-selectin and release of von Willibrand factor. Antibodies against P-selectin (CD62) did not affect XO-mediated neutrophil adherence under static conditions but decreased both rolling and firm adhesive interactions under conditions of shear. We conclude that extracellular XO associates with the endothelium and promotes neutrophil-endothelial cell interactions through dual intercellular adhesion molecule-1 and P-selectin ligation, by a mechanism that involves platelet-activating factor and H2O2 as intermediates.

Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin.

The oxygen radical-producing enzyme xanthine oxidase (XO) can promote neutrophil adherence to endothelium. Recognizing that a balance often exists in inflammatory processes, we sought to determine whether XO initiates antiadherent pathways. We found that bovine pulmonary arterial endothelial cells (EC) exposed to XO released increased amounts of nitrite into the media, reflecting an increased production of nitric oxide (NO). When EC were subjected to shear stress, treatment with XO and/or the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA) increased neutrophil rolling behavior and firm neutrophil adherence to EC in an additive fashion. Both rolling and adherent interactions were abolished by monoclonal antibodies directed against P-selectin. In addition, treatment of EC with XO and/or L-NNA increased both surface expression of P-selectin and release of von Willebrand factor into media. Finally, treatment of EC with the NO donor sodium nitroprusside decreased XO-mediated neutrophil rolling and adherence. We conclude that XO stimulates EC to produce NO and that NO decreases the P-selectin-dependent neutrophil adhesion initiated by XO. Such increases in endogenous NO may constitute an important negative-feedback response to the acute proadhesive effects of XO.

Nitric oxide decreases lung injury after intestinal ischemia.

After injury to a primary organ, mediators are released into the circulation and may initiate inflammation of remote organs. We hypothesized that the local production of nitric oxide (NO) may act to limit the spread of inflammation to secondarily targeted organs. In anesthetized rats, 30 min of intestinal ischemia followed by 2 h of reperfusion (I/R) did not increase lung albumin leak. However, after treatment with NG-nitro-L-arginine methyl ester (L-NAME), intestinal I/R led to increased lung leak, suggesting a protective effect of endogenous NO. The site of action of NO appeared to be the lung and not the gut because 1) after treatment with L-NAME, local delivery of NO to the lung by inhalation abolished the increase in intestinal I/R-induced lung leak; 2) L-NAME had no effect on epithelial permeability (51Cr-labeled EDTA clearance) of reperfused small bowel; and 3) after treatment with L-NAME, local delivery of NO to the gut by luminal perfusion did not improve epithelial permeability of reperfused intestines. Furthermore, L-NAME increased, and inhaled NO decreased, the density of lung neutrophils in rats subjected to intestinal I/R, and treatment with the selectin antagonist fucoidan abolished L-NAME-induced lung leak in rats subjected to intestinal I/R. We conclude that endogenous lung NO limits secondary lung injury after intestinal I/R by decreasing pulmonary neutrophil retention.

Endothelial cell associated anti-elastolytic activity.

Addition of cultured and then carefully-washed bovine pulmonary artery endothelial cells (EC) decreased (p < 0.05) human neutrophil elastase activity (HNE) in vitro. HNE activity was also decreased (p < 0.05) by addition of histone or protamine treated EC. However, addition of papain or trypsin treated EC decreased HNE activity less than addition of untreated cells suggesting that a protein rather than a difference in cell surface charge was responsible. Other observations suggest that EC anti-elastolytic activity was not due to binding of antiprotease from culture media but was dependent on EC protein synthesis. First, addition of EC grown previously in serum-free media decreased HNE activity the same (p < 0.05) as addition of EC cultured in media containing serum. Second, addition of EC treated beforehand with cycloheximide decreased HNE activity less than (p < 0.05) addition of untreated control EC. We conclude that EC most likely make and have anti-elastolytic activity on their surfaces and speculate that EC associated anti-elastolytic activity may modulate inflammatory, repair and other biologic processes involving neutrophil elastase.

Hemorrhage activates NF-kappa B in murine lung mononuclear cells in vivo.

Hemorrhage rapidly increases the expression of proinflammatory and immunoregulatory cytokines in the lungs. Binding elements for the nuclear transcriptional regulatory factors (NF)-kappa B and NF-IL6 (C/EBP beta) are present in the promoter regions of multiple cytokine genes, including those whose expression is increased after blood loss. In the present experiments, we found increased activation in vivo of NF-kappa B in lung mononuclear cells, but not in splenocytes, taken from mice 1 h after hemorrhage. In contrast, hemorrhage did not activate NF-IL6 in lung cells or splenocytes. Inhibition of xanthine oxidase by prior feeding of a tungsten-enriched diet prevented hemorrhage-induced activation in lung cells of NF-kappa B. Incubating splenocytes in vitro with xanthine oxidase activated NF-kappa B but not NF-IL6. Xanthine oxidase-induced activation of NF-kappa B was inhibited by manganese superoxide dismutase, but not by catalase. These results suggest that xanthine oxidase-mediated superoxide anion-dependent activation of NF-kappa B occurs in vivo and in vitro. This mechanism may contribute to increased lung cytokine responses after hemorrhage.

Hypoxia-reoxygenation increases O2-. efflux which injures endothelial cells by an extracellular mechanism.

The mechanisms by which superoxide anion (O2-.) injures reoxygenated vascular cells are not clearly understood. We hypothesized that O2-. formed in an intracellular compartment during reoxygenation may egress through plasmalemmal anion channels and mediate injury from an extracellular site. Bovine pulmonary artery endothelial cells (EC) kept hypoxic for 48 h had increased release of preloaded 51Cr upon reoxygenation. Evidence for an extracellular site of injury was the following. First, decreasing extracellular O2-. levels (measured by cytochrome c reduction) with the anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) leads to decreased 51Cr leak. In contrast to its effect on extracellular O2-., DIDS increased intracellular O2-. levels (measured by nitroblue tetrazolium reduction) following reoxygenation. Second, treatment with exogenous superoxide dismutase (SOD), while having no significant effect on intracellular O2-. levels, also decreased 51Cr leak. Furthermore, cotreatment of EC with DIDS did not abrogate the protective effects of exogenous SOD, suggesting that SOD decreased injury by decreasing extracellular and not intracellular O2-. Finally, exposure of EC to extracellularly generated O2-. (xanthine oxidase/hypoxanthine system) caused injury, which was decreased by SOD but not by blockade of O2-. entry with DIDS. The mechanism by which O2-. injures EC may involve generation of .OH by surface-associated iron, since iron chelators and .OH scavengers of varying membrane permeability all decreased 51Cr release to a similar extent. Furthermore, the iron chelators and .OH scavengers also decreased EC 51Cr leak following exposure to exogenous xanthine oxidase/hypoxanthine but not following exposure to a O2(-.)-independent agent (A23187). We conclude that hypoxia-reoxygenation injures EC in a manner that is at least in part dependent on the efflux of O2-. into the extracellular space. Endogenous and exogenous strategies for protection against reoxygenation injury must target extracellular O2-. as a potentially harmful species.

Sarcoidosis presenting with prolonged fever in a patient with sickle cell anemia.

Although the leading causes of fever and pulmonary infiltrates in sickle cell patients are acute bacterial pneumonia and the acute chest syndrome, the former is usually responsive to antibiotics and not protracted, and the clinical course of the latter is short and self-limited. A patient with sickle cell anemia presented with fever and pulmonary infiltrates of several weeks duration that were unresponsive to antibiotics. Gross endobronchial abnormalities were noted on bronchoscopy, and the biopsies were consistent with sarcoidosis. The diagnosis of sarcoidosis can be difficult to establish in a patient with sickle cell anemia and protracted fever since both disorders are associated with chronic interstitial changes on the chest radiograph as well as restrictive physiology.

Mechanisms of immature myocardial tolerance to ischemia: phenotypic differences in antioxidants, stress proteins, and oxidases.

BACKGROUND: Previous work has suggested tolerance to ischemic injury in newborn myocardium. Although various mechanisms for this protection have been proposed, a link between oxidant-antioxidant factors, stress protein expression, and protection from cardiac ischemia/reperfusion (I/R) injury has not been made in newborn myocardium. We hypothesized total newborn myocardial resistance to I/R is related to decreased oxygen radical producing potential, increased free radical scavenging capacity and augmented stress protein expression. The purposes of the study were to examine in newborn and adult rat hearts (1) functional recovery from I/R, (2) catalase and xanthine oxidase (XO) activities, and (3) heat shock protein 72 (HSP 72) expression. METHODS: Isolated rat hearts (7 to 10 days versus 60 days) were perfused on a nonworking Langendorff apparatus at 60 mm Hg (Krebs-Henseleit buffer, pH 7.4, 37 degrees C) and subjected to 20 minutes of global ischemia and 40 minutes of reperfusion. Left ventricular developed pressure was recorded by using a left ventricular catheter. Catalase and XO were measured by means of standard assays, and HSP 72 was assessed with in situ immunohistochemistry. RESULTS: Newborn rat hearts had greater percentage functional recovery of left ventricular developed pressure after I/R (66.0% +/- 4.2% versus 44.3% +/- 3.5%; p < 0.05). The newborn myocardium also had increased catalase activity (1027.9 +/- 20.6 units/gm versus 707.3 +/- 38.7 units/gm; p < 0.05), whereas the activity of XO was decreased relative to the adult (0.23 +/- 0.01 mU/gm versus 7.6 +/- 1.4 mU/gm; p < 0.05). Furthermore, the expression of HSP 72 was greater in the newborn than the adult control. CONCLUSIONS: Relative to adult hearts, newborn rat hearts are more tolerant to a global ischemic insult followed by reperfusion. This improved functional recovery is associated with decreased oxidant production potential (XO), increased scavenging capacity (catalase), and augmented stress protein expression (HSP 72).

Xanthine oxidase contributes to lung leak in rats subjected to skin burn.

We found that rats subjected to thermal skin injury (skin burn) had increased serum xanthine oxidase (XO) activities, increased serum complement activation (decreased serum CH50 levels), increased erythrocyte (RBC) fragility, increased lung neutrophil accumulation, and increased lung leak compared to sham-treated rats. Treatment of rats with allopurinol (an XO inhibitor) not only decreased serum XO activity, but also decreased complement activation, RBC fragility, lung neutrophil accumulation, and lung leak abnormalities in rats subjected to skin burn. We conclude that XO may contribute to acute lung injury and a number of events associated with the development of acute lung leak following skin burn.