Cigarette smoke condensate inhibits ENaC alpha-subunit expression in lung epithelial cells.

Cigarette smoke has been associated with lung fluid accumulation and increased risk of acute respiratory distress syndrome. It was postulated that ENaC alpha-subunit, which plays a critical role in lung fluid absorption, is affected by cigarette smoke. Cigarette smoke condensate (CSC) was used to treat a human lung epithelial cell line. ENaC alpha-subunit expression was measured using immunoblotting, quantitative PCR and promoter-reporter assays. The current authors found that CSC, without affecting cell survival, suppressed alpha-subunit expression at the transcriptional level in a dose- and time-dependent fashion. This suppression is neither related to nicotine nor due to an increase of hydrogen peroxide levels in CSC-treated cells. CSC also suppressed alpha-subunit core promoter activity. Dexamethasone, which activates the core promoter, was able to attenuate the inhibitory effect of CSC. However, in the presence of CSC, dexamethasone was unable to elicit a full-scale activation of alpha-subunit expression. This inhibition of dexamethasone was partially reversed by withdrawal of CSC. The present results demonstrate that cigarette smoke condensate inhibits ENaC alpha-subunit expression at the transcriptional level through its promoter. This inhibition could be reversed by dexamethasone. The results also suggest that higher doses of dexamethasone may be needed to activate alpha-subunit expression in smokers' lungs compared with nonsmokers' lungs, and that quitting smoking might improve the effectiveness of dexamethasone.

Tumor necrosis factor-alpha-induced activating protein-1 activity is modulated by nitric oxide-mediated protein kinase G activation.

We tested the hypothesis that protein kinase (PK)G activation in response to nitric oxide ((*)NO) mediates tumor necrosis factor (TNF)-alpha-induced activation of the transcription factor activating protein-1 (AP-1) in pulmonary microvessel endothelial monolayers (PEM). The DNA-binding activity of AP-1 was assessed using the electrophoretic mobility shift assay. TNF treatment (1,000 U/ml) for 4 h induced a significant increase in DNA binding of AP-1. The effects of TNF were prevented by the superoxide radical scavenger superoxide dismutase (SOD) (100 U/ml), the (*)NO synthase inhibitor aminoguanidine (100 microM), the guanylate cyclase inhibitor ODQ (100 microM), and the PKG inhibitors KT5823 (1 microM) and 8-bromo-cyclic guanosine monophosphate (cGMP)-thioate (100 microM). Spermine-NO (1 microM) and L-arginine (400 microM) prevented the aminoguanidine-induced ablation of AP-1 activation in response to TNF. Phosphorylation of H-Arg-Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg-OH (BPDEtide), a specific substrate for PKG, measured the activity of cGMP-dependent protein kinase (PKG). TNF for 0.5 h induced an increase in PKG activity that was prevented by aminoguanidine, ODQ, KT5823, and 8-bromo-cGMP-thioate; however, SOD had no effect. The PKG agonist 8-bromo-cGMP (100 microM), when given alone, increased PKG activity but induced significant DNA-binding activity of AP-1 only when given in the ODQ + TNF Group. SIN-1 (1 mM, a peroxynitrite agonist) increased DNA-binding activity of AP-1. SOD prevented SIN-1-induced AP-1 activation, a response similar to that of the SOD + TNF Group. PEM were transfected with the chloramphenicol acetyltransferase (CAT) reporter plasmid pBLCAT2, which contains a regulation sequence responsive to AP-1. The pharmacologic profile of TNF-induced CAT activity was identical to TNF-induced DNA binding by AP-1. Thus, TNF-induced AP-1-dependent gene transcription is modulated by (*)NO-dependent mediated activation of PKG.

Chronic necrotizing pulmonary aspergillosis: approach to management.

OBJECTIVE: To describe our experience with 6 patients and to review the current literature to update the approach to the diagnosis and treatment of chronic necrotizing pulmonary aspergillosis. DESIGN: Patient reports and MEDLINE review of English-language literature published after 1980. RESULTS: Chronic necrotizing pulmonary aspergillosis (CNPA) is a subacute infection most commonly seen in patients with altered local defense from preexisting pulmonary disease or in patients with risk factors that alter systemic immune status. Delays in diagnosis are common. Although initial reports advocated intravenous amphotericin B, itraconazole has emerged as a better initial therapy because of its documented efficacy and minimal toxicity. The dose and duration of therapy should be based on clinical response. In patients who do not respond to medical therapy, pulmonary resection can be considered, but postoperative morbidity is high. Recurrent or relapsing infections occur; chronic maintenance therapy with itraconazole can be considered in patients with residual parenchymal scarring. A wide range of mortality rates has been reported for CNPA. Outcome is most likely influenced by severity of comorbid conditions, extent of underlying pulmonary disease, delays in diagnosis, and initiation of effective therapy.

Endothelial barrier dysfunction and p42 oxidation induced by TNF-alpha are mediated by nitric oxide.

We tested the hypothesis that nitric oxide (.NO) mediates tumor necrosis factor-alpha (TNF-alpha)-induced alterations in permeability and actin of pulmonary artery endothelial monolayers (PAEM). The permeability of PAEM was assessed by the clearance rate of albumin labeled with Evans blue dye. The PAEM Triton-soluble ("cytoskeletal-nonassociated") and -insoluble ("cytoskeletal-associated") lysates were analyzed by Western blot for actin and oxidized protein using polyclonal antibodies to the COOH terminus of actin and dinitrophenylhydrazone (DNP), respectively. PAEM were incubated with TNF-alpha (100 U/ml) for 4 h. Incubation of PAEM with TNF-alpha resulted in increases in 1) the .NO oxidation product nitrite (NO2-), 2) nitrotyrosine immunofluorescence, 3) the oxidation of p42 (tentatively identified as actin), and 4) permeability to Evans blue dye-albumin. The .NO synthase inhibitor aminoguanidine (100 microM) prevented the TNF-alpha-induced increase in NO2-, nitrotyrosine immunofluorescence, oxidized p42, and permeability. Coincubation with L-arginine (200 microM) or the .NO mimic spermine-NO (1 microM) prevented the ablation of the response to TNF-alpha by aminoguanidine. The data indicate that TNF-alpha-induced increases in endothelial permeability and oxidized protein are mediated by .NO in PAEM.

Nitrovasodilator repletion increases TNF-alpha-induced pulmonary edema.

We tested the hypothesis that nitrovasodilator repletion enhances tumor necrosis factor-alpha (TNF-alpha) -induced pulmonary edema. Lungs were isolated from control guinea pigs or 4 h after the intraperitoneal injection of TNF-alpha (1.60 x 10(5) U/kg). In the control and TNF-alpha-injected lungs, the thromboxane mimetic U-46619 (155 pmol/min) caused increases in pulmonary capillary pressure (Ppc) and lung weight (delta W). In control lungs, the nitrovasodilator agonist S-nitroso-N-acetyl-penicillamine (SNAP, 1 microM) attenuated the U-46619-induced increases in Ppc. In lungs injected with TNF-alpha, SNAP had no effect on Ppc and increased delta W. The peroxynitrite (ONOO-) scavenger urate (35 mM) prevented the TNF-alpha +SNAP-induced increases in Ppc and delta W. In addition, chemically synthesized ONOO- (4.0 mM) enhanced U-46619-induced increases in delta W. The data indicate that nitric oxide repletion enhances TNF-alpha-induced pulmonary edema, possibly via ONOO-.

TNF-alpha induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C.

We tested the hypothesis that tumor necrosis factor-alpha (TNF) induces a peroxynitrite (ONOO-)-mediated depletion of glutathione via a protein kinase C (PKC)-dependent mechanism in pulmonary artery endothelial monolayers (PAEM). PAEM were incubated with TNF (1,000 U/ml) for 6 and 18 h. The PAEM were assayed for ONOO(-)-dependent changes in the concentration of luminol, free glutathione [Gfree; i.e., reduced glutathione and oxidized glutathione (GSSG)] and GSSG. TNF treatment decreased luminol and Gfree, and increased GSSG and GSSG/Gfree, compared with treatment with control media. The TNF-induced effects were prevented by co-incubation with the nitric oxide synthase inhibitors NG-monomethyl-L-arginine (1 mM), NG-nitro-L-arginine methyl ester (1 mM), or NG-nitro-L-arginine (1 mM). In addition, the TNF-induced effects were prevented by superoxide dismutase (10 U/ml), which removes O2-, and by urate (0.5 mM) and L-cysteine (3 mM), putative scavengers of ONOO-. The treatment of PAEM with the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) induced similar alterations in luminol and glutathione as TNF. TNF and PMA induced a protein of similar molecular weight (approximately 90 kDa) in the focal contact-rich fraction of PAEM lysate. TNF- and PMA-induced effects were prevented with the specific PKC inhibitor calphostin C (1 microM). The data indicate that TNF-induced PKC activation mediates ONOO- generation, which results in the oxidation and depletion of glutathione in PAEM.

Activation of protein kinase C mediates altered pulmonary vasoreactivity induced by tumor necrosis factor-alpha.

We postulated that tumor necrosis factor-alpha (TNF) "primes" the lung for the development of pulmonary vasoconstriction and edema by activating protein kinase C (PKC). Guinea pigs were injected with TNF (1.6 x 10(5) U/kg i.p.), and the lungs were isolated 4 h later. Compared with controls, TNF pretreatment resulted in greater increases in pulmonary vascular resistance and pressure and lung weight, in response to the thromboxane A2 mimetic, U-46619 (122 pmol/min). Treatment with TNF resulted in 1) pulmonary arterial endothelial PKC activation, 2) increased lung polymorphonuclear neutrophil (PMN) sequestration, 3) increased levels of superoxide radical (O2.) in lung effluent, and 4) decreased nitrite levels (NO2-, oxidation product of nitric oxide) in lung effluent. Intraperitoneal treatment with calphostin C (3 microM, 15 min prior to treatment with TNF) prevented the effects of TNF on 1) PKC activation, 2) the hemodynamic responses to U-46619, and 3) the levels of NO2- and O2(.). PKC activation does not mediate TNF-induced lung sequestration of PMN, since calphostin C had no effect on lung myeloperoxidase activity. The data suggest that PKC activation mediates TNF-induced 1) increases in O2., 2) decreases in NO2-, and 3) increases in vasoreactivity and edema in response to U-46619.

Tumor necrosis factor-alpha decreases pulmonary artery endothelial nitrovasodilator via protein kinase C.

We postulated that tumor necrosis factor-alpha (TNF) decreases endothelium-derived nitrovasodilator(s) via a protein kinase C (PKC)-dependent pathway. Calf pulmonary artery endothelial monolayers (PAEM) were treated with TNF (10, 100, and 1,000 U/ml) for 15 min or 18 h during an 18-h incubation. At the end of the incubation, the cell lysate and supernatant were harvested. Compared with controls, an 18-h incubation with TNF (100 and 1,000 U/ml) resulted in a decrease in NO2- [the oxidation product of nitric oxide (NO)] in PAEM lysate and supernatant. TNF (100 U/ml) treatment for 15 min did not suppress NO2- levels. The decrease in NO2- and the increase in lipid peroxides in response to TNF were prevented by pretreatment (15 min prior to and throughout the incubation) with either calphostin C (1 microM; a specific PKC inhibitor) or the antioxidants N-acetylcysteine (1 mM), 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) (10 mM), and superoxide dismutase (10 U/ml). Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM for 15 min), an activator of PKC, decreased NO2- similarly to TNF. Pretreatment with calphostin C or N-acetylcysteine prior to TNF (10 U/ml) revealed an increase in NO2- levels above control treatment. Treatment with the NO synthase antagonists NG-monomethyl-L-arginine (1 mM) and N-nitroso-L-arginine (1 mM) induced an L-arginine (1 mM)-dependent decrease in NO2- in control but not in TNF-treated PAEM. The induction of NO2- by calcium ionophore (A23187; 500 nM) was not affected by treatment with TNF.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor-alpha alters pulmonary vasoreactivity via neutrophil-derived oxidants.

We postulated that tumor necrosis factor-alpha (TNF) "primes" the lung for the development of pulmonary vasoconstriction and edema by inducing the release of polymorphonuclear leukocyte (PMN)-derived reactive oxidant species (ROS). Guinea pigs were injected with TNF (1.6 x 10(5) U/kg ip), and the lungs isolated 18 h later. Compared with controls, TNF pretreatment resulted in 1) greater increases in lung weight and capillary pressure in response to the thromboxane A2 mimetic U-46619 (365 pmol/min) and 2) an increase in the dose of acetylcholine (ACh) causing 50% of maximal dilation (EC50). The vascular effects of TNF were associated with 1) decreased lung effluent nitrite (NO2-, oxidation product of nitric oxide), 2) increased lung effluent superoxide (O2-), and 3) increased lung myeloperoxidase (MPO). Superoxide dismutase (SOD, 10 U/ml) prevented 1) the effects of TNF on the hemodynamic responses to U-46619 and ACh and 2) the TNF-induced decrease in NO2-. The effects of TNF on lung MPO and effluent O2- were prevented using cyclophosphamide intraperitoneally (100 mg/kg 5 days before, and 50 mg/kg 1 day before, treatment with TNF or control). The data suggest that ROS generated from PMN mediate the decrease in nitric oxide and altered pulmonary vasoreactivity induced by TNF.

Tumor necrosis factor-alpha activates pulmonary artery endothelial protein kinase C.

We investigated the hypothesis that tumor necrosis factor-alpha (TNF) activates pulmonary endothelial protein kinase C (PKC). Confluent bovine pulmonary artery endothelial monolayers were exposed to recombinant human TNF, and the translocation of PKC, an indicator of enzyme activation, was studied using both slot immunoblotting and immunofluorescence. For slot immunoblot analysis, membrane and cytosol lysate fractions were prepared, and PKC antigen was assessed using MC5 monoclonal anti-PKC antibody. TNF (1,000 U/ml for 15 min) induced translocation of PKC into the membrane. Immunofluorescence analysis with the MC5 antibody was also used. Monolayers treated with culture medium showed diffuse cytoplasmic fluorescence. In contrast, treatment with either TNF (1,000 U/ml for 15 min) or 1,2-dioctanoylglycerol (4 x 10(-5) M for 5 min), a diacylglycerol that activates PKC, resulted in translocation of fluorescence to the cell periphery; fine, punctate PKC-associated fluorescence was localized to the margins of cells. The TNF-induced translocation of PKC was inhibited using either IP-300 polyclonal anti-TNF antibody (indicating that the TNF effect was not due to the vehicle or contaminating endotoxin) or calphostin C (10(-6) M for 15 min), which inhibits PKC activation by interacting with the regulatory diacylglycerol-binding domain. TNF treatment had no effect on either the content of PKC, or of total protein, in the membrane + cytosol, and cycloheximide (40 microM for 5 min) did not alter the translocation of PKC induced by TNF; these results indicate that the effect of TNF on PKC translocation was related to neither de novo membrane synthesis of PKC (as opposed to translocation per se) nor nonspecific augmentation of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

TNF-alpha augments pulmonary vasoconstriction via the inhibition of nitrovasodilator activity.

We tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) increases pulmonary vasoconstriction by decreases in nitric oxide- (NO) dependent vasodilation. Lungs were isolated from guinea pigs 18 h after intraperitoneal injection of either TNF-alpha (1.60 x 10(5) U/kg) or control. U-46619 (365 mM/min) caused increases in pulmonary arterial and capillary pressures, pulmonary arterial and venous resistances, and lung weight. TNF-alpha augmented the U-46619-induced increases in pulmonary arterial and capillary pressures, pulmonary arterial and venous resistances, and lung weight. Methylene blue (1 microM), which inhibits the activation of soluble guanylate cyclase by NO, had an effect similar to TNF-alpha on the pulmonary response to U-46619 alone but was not additive to the effect of TNF-alpha. NG-monomethyl-L-arginine (270 microM), an inhibitor of NO generation, also enhanced the response to U-46619. Lung effluent levels of nitrite, the oxidation product of NO, were reduced after treatment with either TNF-alpha or NG-monomethyl-L-arginine compared with U-46619 alone. In addition, lungs isolated after TNF-alpha treatment showed decreased vasodilation in response to acetylcholine (10(-8)-10(-5) M) compared with control; however, vasodilation in response to L-arginine (10 mM) and nitroprusside (10(-6.3) and 10(-6) M), agents that promote NO release, was not decreased in TNF-alpha-treated lungs. The data indicate that TNF-alpha induces an increase in vascular constriction in response to U-46619 and a decrease in vasodilation in response to acetylcholine. The mechanism for the TNF-alpha-induced alteration in pulmonary vascular reactivity may be decreased generation of NO.

Dextran sulfate and heparin sulfate inhibit platelet-activating factor-induced pulmonary edema.

We tested the hypothesis that dextran sulfate and heparin sulfate inhibit platelet-activating factor- (PAF) induced pulmonary edema in the isolated perfused guinea pig lung via a charge-dependent mechanism. Dextran sulfate prevented the changes in pulmonary capillary pressure (Ppc, 7.8 +/- 0.9 vs. 14.0 +/- 0.7 cmH2O), lung weight gain (dW, +0.48 +/- 0.29 vs. +8.41 +/- 2.07 g), and pulmonary edema formation or wet-to-dry weight ratio [(W-D)/D, 6.5 +/- 0.3 vs. 13.2 +/- 2.6] occurring 60 min after PAF infusion (10(-11) M) into an isolated lung. The unsulfated form of dextran had no protective effect [Ppc, dW, and (W-D)/D, 11.9 +/- 1.4 cmH2O, +5.33 +/- 2.18 g, and 11.2 +/- 3.2, respectively]. The unrelated anionic compound, heparin sulfate, also inhibited the PAF response [Ppc, dW, and (W-D)/D, 7.0 +/- 0.5 cmH2O, +0.61 +/- 0.32 g, and 6.1 +/- 0.2, respectively], whereas the partially desulfated form of heparin was not effective in inhibiting PAF-induced edema [Ppc, dW, and (W-D)/D, 15.1 +/- 0.7 cmH2O, +6.07 +/- 1.58 g, and 10.0 +/- 1.2, respectively]. When the metachromatic dye crystal violet was used as an indicator of charge interactions, the sulfated compounds interacted with PAF in vitro. The data indicate that PAF-induced pulmonary edema is inhibited by sulfated polysaccharides, possibly via a charge interaction between negatively charged compounds and PAF.

Tumor necrosis factor-alpha primes pulmonary hemodynamic response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine.

We tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) primes the hemodynamic response to the neutrophil agonist N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in lungs isolated from guinea pigs. Lungs were isolated from animals 18 h after injection of TNF-alpha (3.20 x 10(5) U/kg ip). The infusion of FMLP (300 nM) into lungs isolated after the intraperitoneal administration of TNF-alpha resulted in increases in lung weight, lung (wet-dry)-to-dry weight ratio [(wet-dry)/dry wt], pulmonary capillary pressure, lung myeloperoxidase activity and perfusate thromboxane (Tx)B2 levels. Animals pretreated with the maximal possible amount of endotoxin in the TNF-alpha (1.7 pg endotoxin) did not respond to FMLP. WEB-2086 (37 microM), a platelet-activating factor (PAF) receptor antagonist, added to the perfusate attenuated the hemodynamic and TxA2 response to FMLP. Dazoxiben (0.5 mM), a TxA2 synthetase inhibitor, prevented the FMLP effect. Polyethylene glycol (PEG)-catalase (500 U/ml) added to the perfusate did not affect the FMLP response; however, PEG-catalase (10(5) U/kg) given intraperitoneally with the TNF-alpha decreased the synergism induced by TNF-alpha with FMLP. The data suggest that TNF-alpha primes the lung to the effects of FMLP by increasing the population of resident neutrophils in the lung and/or by in vivo oxidant generation. The pulmonary hemodynamic response and lung edema induced by FMLP are mediated by PAF and TxA2.

Dextran sulfate inhibits PMN-dependent hydrostatic pulmonary edema induced by tumor necrosis factor.

We tested the hypothesis that neutrophil sequestration is required for the development of tumor necrosis factor- (TNF) induced neutrophil- (PMN) dependent pulmonary edema. TNF (3.2 X 10(5) U/kg ip) was injected into guinea pigs 18 h before lung isolation. After isolation, the lung was perfused with a phosphate-buffered Ringer solution. Dextran sulfate (mol wt 500,000) prevented the changes in pulmonary capillary pressure (Ppc; 8.5 +/- 0.8 vs. 12.8 +/- 0.8 cmH2O), lung weight gain (dW; +0.240 +/- 0.135 vs. +1.951 +/- 0.311 g), and pulmonary edema formation or wet-to-dry wt ratio [(W - D)/D; 6.6 +/- 0.2 vs. 8.3 +/- 0.5] at 60 min induced by PMN infusion into a TNF-pretreated lung. The unsulfated form of dextran had no protective effect [Ppc, dW, and (W - D)/D at 60 min: 11.9 +/- 0.9 cmH2O, +1.650 +/- 0.255 g, and 7.3 +/- 0.2, respectively], whereas the use of another anionic compound, heparin, inhibited the TNF + PMN response [Ppc, dW, and (W - D)/D at 60 min: 5.6 +/- 0.4 cmH2O, +0.168 +/- 0.0.052 g, and 6.4 +/- 0.2, respectively]. Isolated lungs showed increased PMN myeloperoxidase (MPO) activity compared with control in TNF-treated lungs at baseline and 60 min after PMN infusion. Dextran sulfate, dextran, and heparin inhibited the increase in MPO activity. The data indicate that inhibition of PMN sequestration alone is not sufficient for the inhibition of PMN-mediated TNF-induced hydrostatic pulmonary edema and that a charge-dependent mechanism mediates the protective effect of dextran sulfate.

Mechanisms of pulmonary edema induced by tumor necrosis factor-alpha.

We tested the hypothesis that human recombinant tumor necrosis factor-alpha (TNF) promotes pulmonary edema by neutrophil-dependent effects on the pulmonary vasculature. The isolated guinea pig lung was perfused with phosphate-buffered Ringer's solution with or without human neutrophils. The infusion of neutrophils (9 x 10(6) total) into lungs isolated after the in vivo administration of TNF (3.2 x 10(5) units/kg) resulted in weight gain (+1.951 +/- 0.311 g versus -0.053 +/- 0.053 g in control) and an increase in the lung (wet-dry)-to-dry weight ratio (8.3 +/- 0.5 versus 6.0 +/- 0.2 in control), indicating the formation of pulmonary edema. The neutrophil-dependent pulmonary edema induced by TNF was associated with a combination of increased capillary permeability (capillary filtration coefficient [Kf,c], 0.170 +/- 0.048 g/min/cm H2O/g at 30 minutes versus 0.118 +/- 0.008 g/min/cm H2O/g at baseline) and increased pulmonary capillary pressure (Ppc, 12.8 +/- 0.8 cm H2O at 60 minutes versus 6.0 +/- 0.3 cm H2O at baseline). The Ppc increase was mediated by thromboxane A2 (TXA2) because the TXA2 synthetase inhibitor Dazoxiben (0.5 mM) prevented the effect (Ppc, 6.7 +/- 0.6 cm H2O at 60 minutes with Dazoxiben), and thromboxane B2 (TXB2) levels were increased in the pulmonary venous effluent (5,244 +/- 599 pg/ml at 60 minutes versus 60 +/- 13 pg/ml at baseline). Studies using WEB-2086 (37 microM), a platelet activating factor (PAF) receptor antagonist, indicated that PAF mediated the increased vascular permeability (Kf,c, 0.107 +/- 0.014 g/min/cm H2O/g at 30 minutes using WEB-2086) and, in part, the increased Ppc (Ppc, 8.4 +/- 0.7 cm H2O at 60 minutes using WEB-2086). In addition, alterations of endothelial peripheral actin bands were noted after TNF administration. The data indicate that TNF induces neutrophil-dependent pulmonary edema associated with increased Ppc (mediated by TXA2 and PAF), increased Kf,c (mediated by PAF), and changes in endothelial peripheral actin bands.

Increased endothelial albumin permeability mediated by protein kinase C activation.

We examined the effects of activation of endothelial protein kinase C (PKC) of the endothelial barrier function. Exposure of confluent bovine pulmonary artery endothelial cell monolayers to phorbol 12-myristate 13-acetate (PMA) resulted in concentration-dependent (10(-8)-10(-6) M) increases in PKC activity and in the transendothelial flux of 125I-albumin. Exposure of the endothelium to 1-oleoyl 2-acetyl glycerol (OAG) also increased the transendothelial flux of 125I-albumin in a concentration-dependent manner. Neither 4 alpha-phorbol didecanoate nor 1-mono-oleoyl glycerol, which do not activate PKC, altered permeability. The increase in 125I-albumin permeability induced by PMA was inhibited by 25 microM H7 (a PKC inhibitor), but not by the control compound HA1004 (25 microM). After 16 h of exposure to PMA, 125I-albumin permeability returned to baseline and a significant reduction in cytosolic PKC activity was noted. Further challenge with PMA at this time resulted in no significant increase in PKC activity indicating downregulation of the enzyme; moreover, this PMA challenge did not increase endothelial permeability. Exposure of endothelial monolayers to phospholipase C (PLC), which increases membrane phosphatidylinositide turnover, or to alpha-thrombin also induced concentration-dependent activation of PKC and increases in 125I-albumin endothelial permeability. The thrombin- and PLC-induced permeability increases were inhibited by H7, but not by HA1004. The activation of endothelial PKC directly by PMA or OAG and by PLC and alpha-thrombin increases the transendothelial albumin permeability, indicating that PKC activation is an important signal transduction pathway by which extracellular mediators increase endothelial macromolecular transport.

Mechanisms of pulmonary edema induced by a diacylglycerol second messenger.

We investigated the effect of dioctanoylglycerol (DOG), a second messenger of protein kinase C (PKC) activation, in the absence and presence of neutrophils in isolated perfused guinea pig lung. DOG was given after a base-line isogravimetric steady-state period. Pulmonary capillary pressure (Ppc) and change in lung weight (delta W) were monitored at 15, 30, and 60 min. Capillary filtration coefficient (Kf,c, an index of vascular permeability) was measured during base-line period and at 30 min. DOG increased the Ppc and delta W at 30 and 60 min, and the Kfc at 30 min. Monooctanoylglycerol, a monoacylglycerol that does not activate PKC, had no effect on Ppc, Kf,c, and delta W. Pretreatment with two different PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine or staurosporin, prevented the pulmonary response to DOG. With neutrophils present, DOG caused greater increases in delta W and the (wet-dry)-to-dry wt ratio compared with DOG group. Response to DOG+ neutrophils was due to oxygen radical production because it was prevented by pretreatment with catalase and because DOG increased superoxide release from neutrophils. PKC activation using DOG in the isolated lung results in pulmonary edema mediated by increases in capillary pressure and vascular permeability. Lung weight-gain response to DOG is greater in the presence of neutrophils. Response to DOG+ neutrophils is mediated by oxygen radicals.

Macrophages activated by fibrin increase albumin permeability across pulmonary artery endothelial monolayers.

We have examined the effects of alveolar macrophages (AM) obtained after challenge with alpha-thrombin on 125I-labeled albumin permeability across ovine pulmonary artery endothelial monolayers. AM were obtained by bronchoalveolar lavage before and after challenging the sheep with alpha-thrombin (80 U/kg). Post-thrombin AM increased 125I-labeled albumin transendothelial permeability, whereas resting AM had no effect (82.7 +/- 7.9% increase versus 17.2 +/- 1.6% increase at the AM:endothelial cell ratio of 1:1; p less than 0.001). The permeability increase was also seen at AM:endothelial cell ratios of 0.2:1 and 5:1. Endothelial permeability to 125I-labeled albumin did not increase after in vitro incubation of macrophages with 10(-8) M thrombin, suggesting that AM are activated as a result of thrombin-induced fibrin microembolism rather than by the alpha-thrombin per se. The increase in permeability was not due to endothelial lysis since macrophages did not cause release of endothelial lactate dehydrogenase. Adherence of AM to the endothelium did not correlate with the ability of AM to increase endothelial permeability. Superoxide anion production was increased when post-thrombin AM were exposed to the endothelial monolayers (30.6 +/- 4.2 nmol/10(6) cells/10 min) compared with production by post-thrombin AM in the absence of endothelial cells (2.5 +/- 0.5 nmol/10(6) cells/10 min). The addition of superoxide dismutase (SOD) blunted the permeability increase induced by AM (32.3 +/- 3.9% increase with SOD versus 84.1 +/- 7.1% increase without SOD; p less than 0.001), indicating that superoxide anion is an important mediator of the macrophage-induced increase in endothelial monolayer permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-2 induces pulmonary edema and vasoconstriction independent of circulating lymphocytes.

We investigated the effect of IL-2 in the isolated guinea pig lung perfused with phosphate-buffered Ringer's solution (containing 0.5 g/100 ml albumin and 5.5 mM dextrose) to determine the mechanism of IL-2-induced pulmonary edema. IL-2 (0 to 10,000 U/ml) was added to the perfusate following a 10 min baseline steady-state period. Pulmonary arterial pressure (Ppa), pulmonary capillary pressure (Ppc), and change in lung weight (as a measure of developing pulmonary edema) were recorded at 0, 10, 30, 40, and 60 min. The capillary filtration coefficient (Kf.c), an index of vascular permeability to water, was measured at 30 and 60 min. Infusion of IL-2 increased Ppc (from 3.9 +/- 0.1 cm H2O at baseline to 8.8 +/- 1.1 cm H2O at 60 min for IL-2 at 2000 U/ml, p less than 0.01; and from 3.8 +/- 0.1 cm H2O at baseline to 8.9 +/- 0.6 cm H2O at 60 min for IL-2 at 10,000 U/ml, p less than 0.01. The lung weight also increased (32% at IL-2 concentration of 2000 U/ml, and 26% at IL-2 concentration of 10,000 U/ml) The capillary filtration coefficient did not change with IL-2 infusion. The IL-2 response was prevented using the pulmonary vasodilator, papaverine. The infusion of IL-2 was associated with the generation of thromboxane A2(TxA2) in the effluent perfusate. Inhibition of TxA2 synthetase using Dazoxiben prevented the pulmonary vasoconstriction and edema response to IL-2. In addition, IL-2 had no effect on the transendothelial clearance of 125I-albumin. The results indicate that IL-2 causes pulmonary edema secondary to an increase in Ppc. The response is mediated by IL-2 stimulation of TxA2 generation from the lung.

Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils.

We studied the effects of intravenous infusion of recombinant human tumor necrosis factor type alpha (rTNF-alpha; 12 micrograms/kg) on lung fluid balance in sheep prepared with chronic lung lymph fistulas. The role of neutrophils was examined in sheep made neutropenic with hydroxyurea (200 mg/kg for 4 or 5 days) before receiving rTNF-alpha. Infusion of rTNF-alpha resulted in respiratory distress and 3-fold increases in pulmonary arterial pressure and pulmonary vascular resistance within 15 min, indicating intense pulmonary vasoconstriction. Pulmonary lymph flow (i.e., net transvascular fluid filtration rate) and transvascular protein clearance rate (a measure of vascular permeability to protein) increased 2-fold within 30 min. The increased permeability was associated with leukopenia and neutropenia. The pulmonary hypertension and vasoconstriction subsided but fluid filtration and vascular permeability continued to increase. Sheep made neutropenic had similar increases in pulmonary transvascular fluid filtration and vascular permeability. rTNF-alpha also produced concentration-dependent increases in permeability of 125I-labeled albumin across ovine endothelial cell monolayers in the absence of neutrophils or other inflammatory mediators. The results indicate that rTNF-alpha increases pulmonary vascular permeability to protein by an effect on the endothelium.