Effects of thromboxane synthase inhibition on air emboli lung injury in sheep

We tested the effects of OKY-046, a thromboxane synthase inhibitor, on lung injury induced by 2 h of pulmonary air infusion (1.23 ml/min) in the pulmonary artery of unanesthetized sheep with chronic lung lymph fistula so as to assess the role of thromboxane A2 (TxA2) in the lung injury. We measured pulmonary hemodynamic parameters and the lung fluid balance. The concentrations of thromboxane B2 (TxB2) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) in plasma and lung lymph were determined by radioimmunoassay. Air infusion caused sustained pulmonary hypertension and an increase in pulmonary vascular permeability. The levels of TxB2 and 6-keto-PGF1 alpha in both plasma and lung lymph were significantly elevated during the air infusion. TxB2 concentration in plasma obtained from the left atrium was higher than that from the pulmonary artery at 15 min of air infusion. When sheep were pretreated with OKY-046 (10 mg/kg iv) prior to the air infusion, increases in TxB2 were prevented. The pulmonary arterial pressure, however, increased similarly to that of untreated sheep (1.8 X base line). The increase in lung lymph flow was significantly suppressed during the air infusion. Our data suggest that the pulmonary hypertension observed during air embolism is not caused by TxA2.     

Effects of thromboxane synthase and cyclooxygenase inhibition on PAF-induced changes in lung function and arachidonic acid metabolism.

PAF was administered as an intravenous bolus (0.1 micrograms/kg) to eight chronically instrumented awake sheep. The effects of pretreatment with an inhibitor of cyclooxygenase (meclofenamate) on PAF-induced changes in lung function were compared to those observed with a specific inhibitor of thromboxane synthase (DP1904). Each animal was studied four times in varied order: PAF alone, PAF + DP1904, PAF + meclofenamate, and DP1904 alone. Saline alone (control), DP1904 alone, and meclofenamate alone did not cause changes in any of the measured variables. DP1904 and meclofenamate significantly attenuated the PAF-induced fall in lung compliance, elevation in peak pulmonary artery pressure, and increased lung lymph flow. Both drugs abolished the PAF-induced increases in lung lymph thromboxane B2 concentrations. Meclofenamate, but not DP1904, blocked the rise in lymph 6-keto-PGF1 alpha. Although meclofenamate blocked the rise in lymph PGE2, DP1904 resulted in levels 2.7 times higher than PAF alone. We conclude that: (1) inhibition of thromboxane synthase is as effective as inhibition of cyclooxygenase in attenuating PAF-induced changes in lung function, and (2) thromboxane synthase inhibition results in augmented production of PGE2 following PAF administration in vivo.     

Ras signaling in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H-ras oncogene converted non-tumorigenic, TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF-induced apoptosis. This finding suggested that Ras activation may play a role in TNF-induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF-induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras-transformed 10TEJ cells inhibited TNF-induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF-induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF-sensitive 10TEJ cells, TNF treatment increased Ras-bound GTP in TNF-sensitive L929 cells but not in TNF-resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF-induced Ras-GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF-induced apoptosis in mouse fibroblasts.     

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.     

Role of complement in endotoxin/platelet-activating factor-induced lung injury.

C receptor-1 is a protein involved in the regulation of C3 and C5-convertases. Recombinant human soluble C receptor-1 has recently been produced and shown to reduce infarct size in a rat model of myocardial ischemia/reperfusion injury. The present study aimed to investigate whether recombinant human soluble C receptor-1 exerts any protective effect on pulmonary injury produced in a rodent model of adult respiratory distress syndrome. In this model, Escherichia coli endotoxin (LPS, 0.1 microgram/kg) combined with platelet-activating factor (1 pmol/kg/min over 60 min, n = 10) caused microvascular lung injury characterized by elevation of myeloperoxidase activity, deposition of C3 and C5b-9 on the endothelium of pulmonary vessels, and pulmonary edema. Furthermore, bronchoalveolar lavage revealed increased neutrophil count and elevated protein concentration. These pulmonary responses were associated with elevated serum TNF-alpha. Pretreatment (10 min, i.v.) with recombinant human soluble C receptor-1 at 10 mg/kg (n = 13), but not at 1 mg/kg, prevented the LPS/platelet-activating factor-induced pulmonary edema (p less than 0.01) and changes in the bronchoalveolar lavage fluid cell count (p less than 0.01) and protein concentration (p less than 0.05), and attenuated the deposition of C3 and C5b-9 to lung vessels. There was no effect on lung myeloperoxidase activity and serum TNF-alpha. Also, C depletion by cobra venom factor (500 U/kg, i.v.) eliminated the pulmonary edema and elevated leukocyte count in bronchoalveolar lavage fluid, but had no effect on lung myeloperoxidase activity and serum TNF-alpha. These data suggest that C factors may play an important role in the pathophysiology of adult respiratory distress syndrome.     

Substrate inactivation of lung thromboxane synthase preferentially decreases thromboxane A2 production

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The production of both products was inhibited by 1-benzylimidazole and furegrelate. Multiple additions of PGH2 dramatically reduced the ability of the enzyme to synthesize TXA2, but did not effect the synthesis of HHT. In addition, 1-benzylimidazole did not protect thromboxane synthase from inactivation with multiple additions of PGH2. When the enzyme was incubated with PGH2 in the presence of 1-benzylimidazole, the synthesis of TXA2 was inhibited. When the inhibitor was removed the enzyme had still been inactivated by PGH2 in the presence of 1-benzylimidazole. Thus the substrate inactivation of the enzyme does not require the production of TXA2. Our data suggests that the synthesis of TXA2 and HHT can be differentially inactivated and may occur at different sites on the enzyme.     

Cathepsin B mediates tumor necrosis factor-induced arachidonic acid release in tumor cells

Arachidonic acid (AA) generated by cytosolic phospholipase A2 (cPLA2) has been suggested to function as a second messenger in tumor necrosis factor (TNF)-induced death signaling. Here, we show that cathepsin B-like proteases are required for the TNF-induced AA release in transformed cells. Pharmaceutical inhibitors of cathepsin B blocked TNF-induced AA release in human breast (MCF-7S1) and cervix (ME-180as) carcinoma as well as murine fibrosarcoma (WEHI-S) cells. Furthermore, TNF-induced AA release was significantly reduced in cathepsin B-deficient immortalized murine embryonic fibroblasts. Employing cPLA2-deficient MCF-7S1 cells expressing ectopic cPLA2 or cPLA2-deficient immortalized murine embryonic fibroblasts, we showed that cPLA2 is dispensable for TNF-induced AA release and death in these cells. Furthermore, TNF-induced cathepsin B-dependent AA release could be dissociated from the cathepsin B-independent cell death in MCF-7S1 cells, whereas both events required cathepsin B activity in other cell lines tested. These data suggest that cathepsin B inhibitors may prove useful not only in the direct control of cell death but also in limiting the damage-associated inflammation.     

Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis

Triggering tumor necrosis factor receptor-1 (TNFR1) induces apoptosis in various cell lines. In contrast, stimulation of TNFR1 in L929sA leads to necrosis. Inhibition of HSP90, a chaperone for many kinases, by geldanamycin or radicicol shifted the response of L929sA cells to TNF from necrosis to apoptosis. This shift was blocked by CrmA but not by BCL-2 overexpression, suggesting that it occurred through activation of procaspase-8. Geldanamycin pretreatment led to a proteasome-dependent decrease in the levels of several TNFR1-interacting proteins including the kinases receptor-interacting protein, inhibitor of kappa B kinase-alpha, inhibitor of kappa B kinase-beta, and to a lesser extent the adaptors NF-kappaB essential modulator and tumor necrosis factor receptor-associated factor 2. As a consequence, NF-kappa B, p38MAPK, and JNK activation were abolished. No significant decrease in the levels of mitogen-activated protein kinases, adaptor proteins TNFR-associated death domain and Fas-associated death domain, or caspase-3, -8, and -9 could be detected. These results suggest that HSP90 client proteins play a crucial role in necrotic signaling. We conclude that inhibition of HSP90 may alter the composition of the TNFR1 complex, favoring the caspase-8-dependent apoptotic pathway. In the absence of geldanamycin, certain HSP90 client proteins may be preferentially recruited to the TNFR1 complex, promoting necrosis. Thus, the availability of proteins such as receptor-interacting protein, Fas-associated death domain, and caspase-8 can determine whether TNFR1 activation will lead to apoptosis or to necrosis.     

Ibuprofen attenuates plasma tumor necrosis factor activity during sepsis-induced acute lung injury.

Plasma tumor necrosis factor (TNF) activity, cardiac index, extravascular lung water, systemic and pulmonary arterial pressures, pulmonary vascular resistance index, and arterial PO2 were monitored for 300 min in four groups of anesthetized pigs: saline-infused animals (n = 5), saline-infused animals given ibuprofen (12.5 mg/kg iv) at 0 and 120 min (n = 4), animals infused for 60 min with live Pseudomonas aeruginosa (Ps, 5 x 10(8) organisms/ml at 0.3 ml.20 kg-1.min-1, n = 6), and animals infused for 60 min with Ps plus ibuprofen administered at 0 and 120 min (n = 4). Infusion of Ps induced significant elevations (greater than 4-fold increase in units/ml of TNF by 60 min, P less than 0.05) in plasma TNF activity (L929 cytolysis assay) and alterations (P less than 0.05) in all hemodynamic and pulmonary parameters within 30-60 min. Ibuprofen administration in sepsis significantly decreased peak TNF activity by 2 units/ml and attenuated many of the physiological alterations due to sepsis. These results show that ibuprofen attenuates sepsis-induced injury and that alterations of acute septic insult are correlated with reduced plasma TNF activity in septic animals given ibuprofen.     

Distinct signaling pathways in TRAIL- versus tumor necrosis factor-induced apoptosis

Trimeric tumor necrosis factor (TNF) binding leads to recruitment of TRADD to TNFR1. In current models, TRADD recruits RIP, TRAF2, and FADD to activate NF-kappaB, Jun N-terminal protein kinase (JNK), and apoptosis. Using stable short-hairpin RNA (shRNA) knockdown (KD) cells targeting these adaptors, TNF death-inducing signaling complex immunoprecipitation demonstrates competitive binding of TRADD and RIP to TNFR1, whereas TRAF2 recruitment requires TRADD. Analysis of KD cells indicates that FADD is necessary for Fas-L- or TRAIL- but not TNF-induced apoptosis. Interestingly, TRADD is dispensable, while RIP is required for TNF-induced apoptosis in human tumor cells. TRADD is required for c-Jun phosphorylation upon TNF exposure. RIP KD abrogates formation of complex II following TNF exposure, whereas TRADD KD allows efficient RIP-caspase 8 association. Treatment with TRAIL also induces formation of a complex II containing FADD, RIP, IKKalpha, and caspase 8 and 10, leading to activation of caspase 8. Our data suggest that TNF triggers apoptosis in a manner distinct from that of Fas-L or TRAIL.     

Butylated hydroxyanisole specifically inhibits tumor necrosis factor-induced cytotoxicity and growth enhancement.

The effect of commonly used food antioxidants on recombinant tumor necrosis factor alpha (rTNF-alpha)-induced cytotoxicity, growth enhancement and adhesion has been evaluated. Butylated hydroxyanisole (BHA) and 4-hydroxymethyl-2,6-di-t-butylphenol (HBP) were the only two of nine antioxidants that completely inhibited rTNF-alpha-induced cytotoxicity in L929 and WEHI 164 fibrosarcoma cells. Ethoxyquin, propyl gallate and butylated hydroquinone only partially inhibited rTNF-alpha-induced cytotoxicity, while the antioxidants butylated hydroxytoluene (BHT), alpha-tocopherol, ascorbic acid and thiodipropionic acid had minimal effects. The only difference between the molecular structure of the efficient HBP and the non-efficient BHT, is a hydroxymethyl group instead of a hydroxyl group on the phenolic ring. Neither BHA nor BHT inhibited the activation of NF kappa B after 10 or 60 min challenge with rTNF-alpha in L929 cells. BHA also inhibited rTNF-alpha-induced, but not rIL-1 beta-induced growth enhancement in FS-4 fibroblasts. Further, BHA blocked both rTNF-alpha-induced and rIL-1 beta-induced prostaglandin E2 synthesis in FS-4 fibroblasts. BHA inhibited the rTNF-alpha-induced release of arachidonic acid in both FS-4 and L929 cells, suggesting that BHA inhibits cellular phospholipase(s). Neither alpha-tocopherol nor BHA inhibited rTNF-alpha-induced adhesiveness of human endothelial cells. The results indicate that BHA is a specific and potent inhibitor of rTNF-alpha- and rTNF-beta-induced cytotoxicity, as well as of rTNF-alpha-induced growth enhancement.     

Thromboxane synthase inhibition and cardiopulmonary function during endotoxemia in sheep.

We studied the cardiopulmonary response to endotoxin (lipopolysaccharide, LPS) in sheep with and without the administration of a thromboxane synthase inhibitor, OKY-046. The animals were instrumented for crystalographic dimension analysis of the left ventricle (LV) and for measurement of LV, aortic, left atrial, and pulmonary arterial pressures and cardiac index, as well as lung lymph flow. They received 1.0 micrograms/kg of Escherichia coli LPS with (n = 8) and without (n = 8) OKY-046 (10 mg/kg bolus, then 10 micrograms.kg-1.min-1). OKY-046 prevented the increase of pulmonary arterial pressure and the decrease of cardiac index that occurred during the early phase of endotoxemia. Between 8 and 12 h after LPS, cardiac index increased from 6.8 +/- 0.7 to 8.9 +/- 0.51.min-1.m-2. Concomitantly, the end-systolic pressure-diameter relationship (ESPDR, sensitive myocardial contractility index) significantly decreased from 14.7 +/- 0.6 to 7.7 +/- 0.7. Other indexes of the LV contractility (+dP/dtmax) were also reduced. OKY-046 prevented the decreases of ESPDR and +dP/dtmax. OKY-046 also attenuated the increased lung lymph flow changes seen with LPS.     

Blockade of tumor necrosis factor-induced Bid cleavage by caspase-resistant Rb

Tumor necrosis factor-alpha (TNF) activates caspase-8 to cleave effector caspases or Bid, resulting in type-1 or type-2 apoptosis, respectively. We show here that TNF also induces caspase-8-dependent C-terminal cleavage of the retinoblastoma protein (Rb). Interestingly, fibroblasts from Rb(MI/MI) mice, in which the C-terminal caspase cleavage site is mutated, exhibit a defect in Bid cleavage despite caspase-8 activation. Recent results suggest that TNF receptor endocytosis is required for the activation of caspase-8. Consistent with this notion, inhibition of V-ATPase, which plays an essential role in acidification and degradation of endosomes, specifically restores Bid cleavage in Rb(MI/MI) cells. Inhibition of V-ATPase sensitizes Rb(MI/MI) but not wild-type fibroblasts to TNF-induced apoptosis and stimulates inflammation-associated colonic apoptosis in Rb(MI/MI) but not wild-type mice. These results suggest that Rb cleavage is required for Bid cleavage in TNF-induced type-2 apoptosis, and this requirement can be supplanted by the inhibition of V-ATPase.     

E1A sensitizes cells to tumor necrosis factor-induced apoptosis through inhibition of IkappaB kinases and nuclear factor kappaB activities.

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.     

Effects of dibutyryl cAMP on pulmonary air embolism-induced lung injury in awake sheep

To assess the role of intracellular adenosine 3',5'-cyclic monophosphate (cAMP), we tested the effects of dibutyryl cAMP (DBcAMP), an analogue of cAMP, on lung injury induced by pulmonary air embolism in awake sheep with chronic lung lymph fistula. We infused air (1.23 ml/min) in the pulmonary artery for 2 h in untreated control sheep. In DBcAMP-pretreated sheep DBcAMP was infused (1 mg/kg bolus and 0.02 mg.kg-1.min-1 constantly for 5 h); after 1 h from beginning of DBcAMP administration the air infusion was started. After the air infusion, pulmonary arterial pressure (Ppa) and lung lymph flow rate (Qlym) significantly increased in both groups. DBcAMP-pretreated sheep showed significantly lower responses in Qlym (2.7 X base line) compared with untreated control sheep (4.6 X base line); however, Ppa, left atrial pressure, and lung lymph-to-plasma protein concentration ratio were not significantly different between the two groups. Although plasma and lung lymph thromboxane B2 and 6-ketoprostaglandin F1 alpha concentrations increased significantly during the air infusion, DBcAMP-pretreated sheep showed significantly lower responses. Thus DBcAMP infusion attenuated pulmonary microvascular permeability induced by air embolism. We conclude that pulmonary vascular permeability is in part controlled by the intracellular cAMP level.     

Oleic acid lung injury in sheep

Intravenous infusion of oleic acid into experimental animals causes acute lung injury resulting in pulmonary edema. We investigated the mechanism of oleic acid lung injury in sheep. In experiments with anesthetized and unanesthetized sheep with lung lymph fistulas, we measured pulmonary arterial and left atrial pressures, cardiac output, lung lymph flow, and lymph and plasma protein concentrations. We injured the lungs with intravenous infusions of oleic acid at doses ranging from 0.015 to 0.120 ml/kg. We found that oleic acid caused reproducible dose-related increases in pulmonary arterial pressure and pulmonary vascular resistance, arterial hypoxemia, and increased protein-rich lung lymph flow and extravascular lung water. The lung fluid balance changes were characteristic of increased permeability pulmonary edema. Infusion of the esterified fat triolein had no hemodynamic or lung fluid balance effects. Depletion of leukocytes with a nitrogen mustard or platelets with an antiplatelet serum had no effect on oleic acid lung injury. Treatment of sheep before injury with methylprednisolone 30 mg/kg or ibuprofen 12.5-15.0 mg/kg also had no effects. Unlike other well-characterized sheep lung injuries, injury caused by oleic acid does not require participation of leukocytes.