Introduction

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The acute respiratory distress syndrome (ARDS) affects over 150,000 patients per year in the United States, with a mortality rate in the range of 40% (1). The syndrome follows diverse physiologic insults including overwhelming infection, hemorrhagic shock, aspiration, pancreatitis, and major trauma. Lung injury in ARDS is characterized by an increase in the permeability of the alveolar-capillary membrane, leading to alterations in gas exchange and lung mechanics. Several lines of evidence suggest that the neutrophil is the principal cellular mediator in the development of acute lung injury. For example, experimental neutrophil depletion and strategies designed to limit neutrophil- endothelial interactions prevent the development of acute lung injury in several animal models (2, 3). Furthermore, intravenous or airway instillation of substances known to attract and activate neutrophils, including platelet activating factor (PAF) (4), phorbol myristate acetate (PMA) (5), and complement fragments (6) can produce lung injury resembling that in ARDS.

Neutrophils may cause injury to the alveolar-capillary unit through both nonoxidative and oxidative mechanisms. Support for the former is derived from studies in which either protease or elastase inhibitors have mitigated acute lung injury in experimental animals (7, 8). A role for reactive oxygen species in this process is suggested by the finding of elevated levels of hydrogen peroxide in the expired breath of patients with ARDS (9), as well as by an increase in circulating products of lipid peroxidation (10). Moreover, various antioxidant strategies appear to attenuate acute lung injury in vivo (11, 12). Although the relative contributions of oxidative and nonoxidative mechanisms of tissue injury are unclear, it is evident that activation of pulmonary capillary endothelial cells and alveolar macrophages precedes, and thus contributes to, lung leukosequestration through upregulation of vascular adhesion molecules (13, 14) and elaboration of proinflammatory cytokines (15, 16), respectively.

Traditionally, oxidants have been considered to exert their effects through a direct toxic action on target cells. However, recent studies have suggested a contributory role for oxidants in gene induction. Nuclear factor-B (NF-B) is a pleiotropic transcription factor activated by low levels of reactive oxygen species and inhibited by antioxidants (17). Consensus binding sequences for NF-B have been identified in the promoter regions of several genes implicated in the pathogenesis of ARDS, including those for tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and IL-8 (18), as well as the endothelial adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) (19). Further, increased NF-B binding activity has been reported in alveolar macrophages isolated from patients with ARDS (20). These data suggest that local oxidative stress may play a role in the perpetuation of the local pulmonary inflammatory response through gene induction. Conversely, antioxidants may in part mediate their salutary effects by precluding induction of the cytokine cascade and upregulation of adhesion molecules.

The dithiocarbamates represent a class of antioxidants reported to be potent inhibitors of NF-B in vitro (17). The metal-chelating properties of the diethyl derivative of dithiocarbamate (diethyldithiocarbamate; DDTC) have been exploited for decades for the treatment of metal poisoning in humans (21). More recently, DDTC has been used to retard the onset of acquired immune deficiency syndrome (AIDS) in human immunodeficiency virus (HIV)- infected individuals (22), a phenomenon thought to be related to its effect on NF-B activation (23). In this regard, the most effective NF-B inhibitor appears to be the pyrrolidine derivative of dithiocarbamate (pyrrolidine dithiocarbamate; PDTC) as a result of its ability to traverse the cell membrane and its prolonged stability in solution at physiologic pH (24).

The potential for modulating both cell activation and the effects of oxidants with the dithiocarbamates suggests that these agents may offer therapeutic benefit in acute lung injury. The present studies were designed to evaluate the effectiveness of PDTC in a rodent model of acute lung injury induced by intratracheal challenge with endotoxin, and to investigate the mechanisms underlying its protective effect.

	Materials and Methods

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Reagents

Escherichia coli 0111:B4 lipopolysaccharide (LPS) and powdered Brewer's thioglycollate were obtained from Difco Laboratories (Detroit, MI). Thioglycollate was dissolved in H₂O, autoclaved, and stored in the dark at room temperature until uniformly green and clear. PDTC, pyroglutamic acid (PGA), N-acetylcysteine (NAC), prostaglandin E₁ (PGE₁), PMA, and thiobarbituric acid were all obtained from Sigma Chemical Company (St. Louis, MO). [¹²⁵I]-albumin was obtained from Merck Frosst (Montreal, Quebec). [-³²P]dCTP and [-³²P]ATP were both purchased from Dupont (Boston, MA). Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) balanced salt solution (LBSS) was purchased from Exoxemis (San Antonio, TX). Calcium/magnesium-free Hanks' balanced salt solution (HBSS) was obtained from GIBCO/BRL Laboratories (Grand Island, NY).

Induction of Acute Lung Injury

All animal studies were performed in accordance with guidelines set by the Toronto Hospital Animal Care Committee and the Canadian Council on Animal Care. Lung injury induced by intratracheal challenge with endotoxin has been well characterized and demonstrated to depend on neutrophil influx mediated by upregulation of E-selectin and ICAM-1 on the pulmonary capillary endothelium (13, 25), and induction of TNF- and IL-1 secretion by alveolar macrophages (26, 27). Male Sprague-Dawley rats weighing 250 to 275 g were obtained from Charles River Laboratories (Constante, Quebec). Animals were housed in standard wire-bottom cages, fed standard rat chow and water ad libitum, and allowed to acclimatize before use. Prior to experimentation, animals were fasted overnight and anesthetized with sodium pentobarbital (50 mg/kg) administered intraperitoneally. A tracheostomy was performed and 0.5 ml of saline containing 500 µg of LPS was instilled, followed by 20 mechanically ventilated breaths produced with a rodent ventilator. Sham-treated animals received 0.5 ml of saline alone. Animals were given PDTC, NAC, or saline by intraperitoneal injection at varying time intervals prior to or following LPS challenge. Animals were maintained at 37°C with the use of warming blankets until recovery from anesthesia.

Assessment of Lung Injury

Pulmonary transcapillary albumin transit was assessed by injection of 1 µCi of [¹²⁵I]albumin into the inferior vena cava 30 min prior to killing, as previously described (12). At the end of the experimental protocol, rats were ventilated, heparin (100 U) was injected into the right ventricle, and 1 ml of blood was withdrawn by cardiac puncture. Following exsanguination, lungs were perfused blood-free by cannulating the pulmonary artery and infusing 10 ml of a low-potassium dextran solution containing 0.5 µg/L of PGE₁. The left ventricle, left atrium, and mitral valve were opened widely to allow free drainage of effluent. The left lung and right lower lobe were used to calculate a permeability index (PI) as follows:

(1)

The remaining lung was immediately frozen in liquid nitrogen for total RNA extraction.

Bronchoalveolar Lavage

Bronchoalveolar lavage fluid (BALF) was collected both for cell counting and measurement of TNF-. Forty milliliters of phosphate-buffered saline (PBS; pH 7.4) was instilled via the trachea in 10-ml aliquots and then gently withdrawn. The first 10 ml of the lavage fluid was centrifuged at 400 × g and the cell-free supernatant assayed for TNF- with an enzyme-linked immunosorbent assay (ELISA) (28). The pellets from individual aliquots were combined and cell counts were determined with a Coulter counter.

Lung RNA Extraction and Northern-blot Analysis

Total RNA from lungs was obtained with the guanidium isothiocyanate method (29). Briefly, lungs were harvested from treated animals and immediately frozen in liquid nitrogen. Lungs were then thawed and homogenized in 10 ml of 4 M guanidine isothiocyanate containing 25 mM sodium citrate, 0.5% sarcosyl, and 100 mM -mercaptoethanol. RNA was denatured, electrophoresed through a 1.2% formaldehyde-agarose gel, and transferred to a nylon membrane. Hybridization was done with a [-³²P]dCTP-labeled, random-primed murine TNF-, ICAM-1, or 18S ribosomal subunit complementary DNA (cDNA) probe (30). Messenger RNA (mRNA) expression was quantitated with a phosphoimager and accompanying ImageQuant software (Molecular Dynamics, Sunnyvale, CA), and was standardized to the 18S ribosomal RNA (rRNA) signal to correct for any variability in gel loading.

Evaluation of NF-B Activation by EMSA

Macrophages in peritoneal exudate were collected by peritoneal lavage from rats five days after intraperitoneal injection of 10.0 ml of thioglycollate broth. The resultant cell population consisted of 80 to 90% macrophages as assessed by nonspecific esterase staining, Wright's staining, and transmission electron microscopy. Cell viability was evaluated by trypan blue exclusion, and exceeded 95% in all studies. Nuclear extracts were prepared as follows: cells were washed twice with cold HBSS, pelleted, and resuspended in lysis buffer containing 10 mM 4-(2-hydroxyethyl)-l-piperazine-N'-2-ethanesulfonic acid (Hepes) (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethyl sulfonyl fluoride (PMSF), and 0.1% NP-40. After a 10-min incubation on ice, the lysates were spun at 13,000 rpm at 4°C for 10 min. Supernatants were collected and frozen immediately in dry ice. The nuclear pellet was resuspended in 15 µl/10⁷ cells of nuclear extract buffer containing 20 mM Hepes (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM ethylene diamine tetraacetic acid (EDTA), 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.15 mM spermine, and 5 µg/ml each of leupeptin, pepstatin, and aprotinin. After a 15-min incubation at 4°C, supernatants were centrifuged at 14,000 rpm at 4°C for 15 min, diluted with 75 µl of buffer containing 20 mM Hepes (pH 7.9), 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF, and frozen immediately on dry ice. Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Five micrograms of protein were preincubated with the nonspecific DNA competitor poly (dI-dC) (5 µg; Pharmacia, Piscataway, NJ) for 10 min at room temperature [-³²P]ATP radiolabeled probe containing the NF-B₃ site of the murine TNF- gene promoter with the sequence 5'-CAAACAGGGGGCTTTCCCTCCTC-3' was incubated for an additional 30 min at room temperature. DNA-protein complexes were resolved on a 5% nondenaturing polyacrylamide (60:1 crosslink)/Tris glycine gel, and autoradiographs were prepared by exposure at 70°C, using X-OMAT film (Kodak, Rochester, NY). To demonstrate specificity of the protein-DNA complex, a 125-M excess of unlabeled probe was added to the nuclear extract before adding the radiolabeled probe.

Evaulation of Lipid Peroxidation

The assay for tissue lipid peroxidation depends on the production of malondialdehyde (MDA), a three-carbon degradation product of lipid peroxidation. Detection of lung MDA can be done colorimetrically by evaluating levels of thiobarbituric acid-reactive substances in whole-lung homogenates (31). The assay was performed as previously described, with minor modifications (32). Lungs were removed from animals immediately after killing, and were rinsed with ice-cold saline to remove excess blood. All subsequent steps were done at 0 to 4°C. Following rinsing, lungs were quickly weighed and finely minced. Approximately 1 g of lung sample was homogenized with a Brinkman Polytron in a sufficient volume of ice-cold 50 mM Tris-EDTA buffer (pH 7.4; 3 mM EDTA) to produce a 20% homogenate. Homogenate fractions (1.0 ml) were added to 1% thiobarbituric acid solution. The mixture was then incubated at 110°C for 10 min. The absorbance of the solution was measured at 525 nm and standardized for protein content as determined by the Lowry method (33).

PMN Chemiluminescence

Whole-blood chemiluminescence was assessed as previously described (34). Briefly, 20 µl of blood was obtained via cardiac puncture and added to 400 µl of LBSS. Phorbol ester-stimulated chemiluminescence was followed over a 60-min period with an Automat LB 953 luminometer (Wildbad, Germany). Total chemiluminescence was integrated over this interval with software provided by the manufacturer and standardized to the number of PMN in the sample of whole blood.

Statistical Analysis

Results are expressed as mean ± SEM. Statistical significance among the group means was assessed by one-way analysis of variance (ANOVA). Post hoc testing was done with Bonferroni's modification of the t-test.

	Results

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Intratracheal LPS challenge caused a 15-fold increase in lung microvascular permeability as assessed by the transpulmonary flux of radiolabeled albumin (Figure 1a). Intraperitoneal injection of PDTC (1 mmol/kg) 30 min prior to LPS challenge significantly attenuated lung permeability as compared with that of saline-treated animals receiving LPS. This effect occurred in a dose-dependent manner, with residual efficacy at doses as low as 0.25 mmole/kg (Figure 1b). To determine the optimal time of administration of PDTC, this agent was administered at various times relative to LPS injection. Although PDTC administration either concomitant with or 30 min before LPS challenge conferred maximal protection against increased lung permeability, a significant degree of protection was evident even when PDTC was administered 60 min after LPS administration (Figure 1c). All subsequent studies were done with 1 mmol/kg PDTC administered 30 min before LPS challenge.

View larger version (17K): [in this window] [in a new window]   Figure 1.   (a) Effect of PDTC on lung injury. Pulmonary transcapillary albumin transit was used to assess alterations in lung permeability. Animals were pretreated with PDTC (1 mmole/kg) or saline 30 min prior to challenge with intratracheal endotoxin (500 µg) or vehicle, and were killed 4 h later. Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.001 versus no LPS; P < 0.001 versus LPS, no PDTC. (b) Dose-response effect of PDTC on lung permeability as assessed in (a). Animals were pretreated with PDTC (0 to 1 mmole/kg) 30 min prior to intratracheal endotoxin (500 µg) challenge, and were killed 4 h later. Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.05 versus no PDTC. (c) Evaluation of optimal time of administration of PDTC. Lung permeability was assessed as in (a). Animals were pretreated with PDTC (1 mmole/kg) or saline at varying time points prior to and following intratracheal endotoxin (500 µg) administration, and were killed 4 h later. Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.05 versus no PDTC.

To determine whether the protective effect of PDTC was related to its antioxidant properties, we administered a structurally related analog of PDTC, pyroglutamic acid (PGA), to rats treated with LPS. Both PDTC and PGA contain a pyrrolidine ring, but in PGA a glutamic acid residue replaces the dithiocarbamate moiety, rendering PGA free of any antioxidant properties. As shown in Figure 2a, PGA was ineffective in mitigating LPS-induced lung injury. Furthermore, the degree of protection conferred by PDTC was similar to that produced by high doses of the unrelated thiol-based antioxidant NAC (0.5 g/kg) (Figure 2b). Considered together, these data suggest a beneficial effect of PDTC against LPS-induced lung injury, by virtue of its antioxidant activity. Subsequent studies focused on the possible mechanisms underlying this effect.

View larger version (15K): [in this window] [in a new window]   Figure 2.   (a) Animals were pretreated with PGA (1 mmole/kg) 30 min prior to intratracheal endotoxin (500 µg) challenge and were killed 4 h later. Pulmonary transcapillary albumin transit was used to assess alterations in lung permeability. Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.05 versus no LPS. (b) Animals were pretreated with NAC (0.5 g/kg) 1 h prior to intratracheal endotoxin (500 µg) challenge, and were killed 4 h later. Lung injury was assessed as described in (a). Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.001 versus no LPS; P < 0.05 versus LPS, no NAC.

The proinflammatory cytokine TNF- is responsible for a number of the effects mediated by endotoxin, and has been shown to play an important role in the initiation of lung injury caused by intratracheal LPS (26). Activation of the TNF- gene is dependent on binding of the transcription factor NF-B to its consensus motifs in the TNF- promoter region (35). Through its cell-permeability and potent antioxidant properties, PDTC has been reported to be an effective and specific inhibitor of NF-B activation (17). To determine whether PDTC mediated its protective effect against LPS-induced lung injury through this mechanism, we evaluated activation of the TNF- gene in vivo by assessing whole-lung TNF- mRNA expression and accumulation of TNF- protein in BALF. As demonstrated in Figures 3 and 4, there was a marked increase in lung TNF- mRNA expression and BALF TNF- levels in response to LPS challenge. However, prior treatment with PDTC failed to prevent the induction of TNF- mRNA or protein.

View larger version (11K): [in this window] [in a new window]   Figure 3.   BALF TNF- levels in rats pretreated with PDTC (1 mmole/kg) or saline 30 min before intratracheal endotoxin (500 µg) or vehicle challenge and killed 4 h later. Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.01 versus no LPS.

View larger version (20K): [in this window] [in a new window]   Figure 4.   (a) Northern blot analysis of TNF- mRNA expression in lungs from rats pretreated with PDTC (1 mmole/kg) or saline 30 min before intratracheal endotoxin (500 µg) or vehicle challenge and killed 4 h later. (b) TNF- mRNA expression was quantitated and normalized to 18S rRNA as described in MATERIALS AND METHODS. Data are expressed as mean ± SEM of four animals per group. *P < 0.05 versus no LPS.

In previous studies, the antioxidant NAC was shown to partly lessen IL-1-induced lung injury by reducing lung neutrophil influx (11). Blockade of the endothelial adhesion molecule ICAM-1 has been shown to attenuate pulmonary leukosequestration and lung injury following intratracheal challenge with LPS (25). Since induction of the ICAM-1 gene is NF-B-dependent (19), we postulated that the beneficial effect of PDTC might occur through the ability of this agent to prevent lung neutrophil sequestration. As shown in Figure 5, quantitation of BALF neutrophils showed an equivalent degree of lung neutrophil influx following LPS challenge in saline- and in PDTC-treated animals. Moreover, PDTC did not appear to modulate expression of the ICAM-1 gene, since there was no significant difference in lung ICAM-1 mRNA expression following induction of lung injury with and without pretreatment with this agent (Figure 6).

View larger version (11K): [in this window] [in a new window]   Figure 5.   Quantitation of BALF neutrophils derived from rats pretreated with PDTC (1 mmole/kg) or saline 30 min before intratracheal endotoxin (500 µg) or vehicle challenge and killed 4 h later. Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.01 versus no LPS.

View larger version (25K): [in this window] [in a new window]   Figure 6.   (a) Northern blot analysis of ICAM-1 mRNA expression in lungs from rats pretreated with PDTC (1 mmole/kg) 30 min before intratracheal endotoxin (500 µg) or vehicle challenge and killed 4 h later. (b) ICAM-1 mRNA expression was quantitated and normalized to 18S rRNA as described in MATERIALS AND METHODS. Data are mean ± SEM of four animals per group. *P < 0.05 versus no LPS. Thee was no significant difference in ICAM-1 MRNA expression in LPS- and LPS-PDTC-treated animals.

Despite its effects in vitro, administration of PDTC in vivo was without a significant effect on induction of the NF-B-dependent gene products TNF- and ICAM-1. To directly evaluate the effects of PDTC on NF-B activation in vivo, we evaluated NF-B DNA binding activity in nuclear extracts of macrophages derived from animals treated with PDTC, using the electrophoretic mobility shift assay (EMSA). Following intratracheal challenge with LPS, the massive neutrophil influx into the alveoli precluded isolation of a sufficiently pure population of alveolar macrophages to evaluate NF-B activation. This problem was overcome by first inducing an influx of macrophages into the peritoneal cavity through the intraperitoneal administration of thioglycollate, then administering either PDTC (1 mmole/kg) or saline by intraperitoneal injection 30 min before intraperitoneal administration of LPS (500 µg). As shown in Figure 7, there was very little constitutive NF-B binding activity in cells derived from control animals, an effect slightly augmented by prior treatment with PDTC. Intraperitoneal challenge with LPS resulted in a marked increase in NF-B binding activity. PDTC pretreatment had no effect on induction of NF-B binding activity, explaining the lack of efficacy of this agent in preventing upregulation of NF-B-dependent gene products.

View larger version (82K): [in this window] [in a new window]   Figure 7.   Effect of PDTC on LPS-induced NF-B DNA binding activity in peritoneal exudate macropahges stimulated with LPS in vivo. Animals were given thioglycollate (10.0 ml) by intraperitoneal injection 5 days prior to experimentation. Thirty minutes before injection of LPS (500 µg intraperitoneally), animals were pretreated with an intraperitoneal injection of either saline or PDTC (1 mmole/kg). Nuclear extracts were prepared from macrophages harvested 3 h after LPS administration, incubated with a [-32P] ATP-labeled oligonucleotide encompassing the murine TNF- NF-B3 consensus motif, and analyzed with the electrophoretic mobility shift assay. Unlabeled competitor oligonucleotide, present at 125 M excess, was used in Lane 5. Data are representative of three separate experiments.

Having demonstrated that PDTC reduces lung injury despite preservation of LPS-induced neutrophil influx, we performed studies to determine whether PDTC attenuated oxidant-mediated injury as a mechanism underlying its protective effect. Peroxidation of membrane phospholipids represents one mechanism by which neutrophil-derived oxidants induce cellular injury. To determine whether PDTC mitigated lung injury by preventing lipid peroxidation, we evaluated levels of MDA, a marker of peroxidation in whole-lung homogenates. In lungs exposed to LPS, there was a significant increase in thiobarbituric acid-reactive substances (Figure 8). This increase was significantly attenuated by pretreatment with PDTC.

View larger version (13K): [in this window] [in a new window]   Figure 8.   Lipid peroxidation levels as measured through the formation of thiobarbituric acid (TBA) reactants. Animals were pretreated with PDTC (1 mmole/kg) or saline 30 min before intratracheal endotoxin (500 µg) or vehicle challenge and killed 4 h later. Data are expressed as mean ± SEM. The numbers of animals per group are indicated in parentheses. *P < 0.001 versus no LPS; P < 0.001 versus LPS, no PDTC.

Injury to the alveolar-capillary unit occurs in part as a direct consequence of polymorphonuclear leukocyte (PMN)- mediated oxidant injury. Therefore, altered PMN activity could be responsible for the observed reduction in injury and lipid peroxidation with PDTC. We postulated that PDTC might exert its protective effects by attenuating the release of reactive oxygen species by neutrophils. This parameter was evaluated by measuring phorbol ester-induced whole-blood chemiluminescence in animals pretreated with PDTC. As shown in Figure 9, PMA induced a marked increase in neutrophil-derived oxidants. PDTC did not impair the phorbol ester-induced chemiluminescent response. Rather, there appeared to be some potentiation. Basal levels (no PMA stimulation) of chemiluminescence were unaffected (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 9.   Peripheral blood neutrophil chemiluminescence as determined through phorbol ester stimulation of whole blood in the presence of the chemiluminigenic probe luminol. Blood was collected from rats pretreated with PDTC (1 mmole/kg) or saline 30 min before intratracheal endotoxin (500 µg) challenge and killed 4 h later. Chemiluminescence was measured every 60 s for a period of 60 min following addition of PMA (12.5 µM). The inset demonstrates chemiluminescence in sham- and LPS-treated animals, integrated over the period of activation and standardized with the peripheral blood neutrophil count. Data are expressed as mean ± SEM of eight animals per group. *P < 0.05 versus LPS, no PDTC.

	Discussion

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The data presented in this report demonstrate that PDTC significantly attenuates endotoxin-induced acute lung injury. The effect persists even when the agent is administered 1 h after endotoxin challenge, and appears to be related to the antioxidant properties of the PDTC molecule. The latter conclusion is based on several lines of evidence. First, PGA, a molecular congener of PDTC without antioxidant properties, was without effect, whereas NAC, a thiol derivative exerting antioxidant activity by a different mechanism, exhibited a protective effect. Second, PDTC blunted the LPS-induced increase in lipid peroxidation, a marker of oxidant-mediated cellular injury. Third, prior treatment with PDTC did not reduce PMN production of oxidants.

On the basis of the well described in vitro effects of antioxidants on gene activation mediated through an inhibitory effect on NF-B (17, 19, 36), we postulated that administration of PDTC might lessen lung injury by preventing induction of genes implicated in the development and perpetuation of the inflammatory response. Specifically, we studied induction of the proinflammatory cytokine TNF- and the vascular adhesion molecule ICAM-1, since both have been implicated in the development of acute lung injury, and induction of their respective genes has been shown to be sensitive to antioxidants in vitro (39, 40). Our data demonstrate that PDTC has no effect on LPS-induced NF-B binding activity or on levels of TNF- and ICAM-1 mRNA in vivo. There may be several reasons for these observations. First, inadequate local concentrations of PDTC may have accounted for its lack of effect on gene activation, although when higher doses (> 2 mmole/kg) were administered, several animals developed neuromuscular irritability and hypersalivation, with the frequency of adverse effects increasing in a dose-dependent fashion. Even when macrophages were directly exposed to high concentrations of PDTC within the peritoneal cavity, no effect on NF-B activation was observed, suggesting that the lack of effect was unlikely to be due to insufficient local concentrations of PDTC.

Alternatively, the failure to detect altered gene activation in vivo may relate to the inherent variability in the ability of antioxidants to produce such activation in vitro, depending on the cell type and species studied. For example, although induction of the ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) genes are NF-B-dependent (19), PDTC appears to downregulate VCAM-1 but not ICAM-1 expression in human endothelial cells (36, 41). By contrast, PDTC completely inhibits human fibroblast ICAM-1 expression under similar conditions (40). Contrasting effects have also been reported in human monocytes and promyelocytic cells when antioxidant modulation of the TNF- gene has been studied (39, 42). Moreover, species specificity may account for some of the contrasting observations, since PDTC potentiates LPS- induced TNF- gene expression in murine macrophages (43), yet attenuates it in human monocytes (39). Beyond this, the complexities of an in vivo system may preclude analysis of a direct effect of antioxidants on gene activation. Cell activation in vitro occurs through a well-defined stimulus, a situation unlikely to be present in vivo, where cells may be activated through several different mechanisms acting simultaneously. As in the present study, these possibilities may have contributed to the ability of antioxidants of the 21-aminosteroid class to reduce hyperoxic lung injury without any effect on gene activation (44).

The absence of any effect on NF-B activation and NF-B-dependent gene products contrasts with recent data suggesting that administration of NAC (0.2 to 1 g/kg) causes a modest reduction in whole-lung NF-B binding activity in acute lung injury induced by the intraperitoneal administration of LPS (45). NAC also mediated a significant reduction in lung PMN influx, an effect similarly reported after the intratracheal administration of IL-1 (11). In the present study we were unable to demonstrate a reduction in neutrophil influx with PDTC. Although we did not evaluate the levels of cytokine-induced neutrophil chemoattractant (CINC), other local factors that may contribute to PMN influx, including TNF- and ICAM-1, were unaffected by PDTC. The disparity may relate to the type or magnitude of the stimulus used to invoke lung injury. The large doses of LPS used in the present study caused a 15-fold increase in lung permeability, whereas lung permeability increased by less than 3-fold following challenge with IL-1 (11). Further, in studies by Blackwell and colleagues, the BAL neutrophil counts were only one-third the values reported in the present study, suggesting a lesser degree of injury. Also supporting the idea that differences in models and treatment may have contributed to the differences in our results and those reported by Blackwell and colleagues (45) was that NAC administration in the present studies had no effect on PMN influx (data not shown), yet prevented lung injury.

Having shown that PDTC did not attenuate lung injury through an effect on cell activation, we evaluated whether this antioxidant reduced lung lipid peroxidation. A decrease in thiobarbituric acid-reactive substances in PDTC-treated animals provided evidence for a reduction in oxidant injury. This was not due to attenuation of neutrophil oxidant production, since there was preservation of oxidative metabolism in neutrophils obtained from animals pretreated with PDTC. PDTC may prevent lipid peroxidation by one of two mechanisms (17). As a direct oxidant scavenger, it limits the availability of superoxide and hydrogen peroxide, both of which are substrates for the subsequent formation of hydroxyl radicals and peroxynitrite, reactive species that are potent initiators of lipid peroxidation (46). PDTC is also a chelator of heavy metals, and in this capacity probably prevents formation of hydroxyl radicals produced through the Haber-Weiss reaction. Consistent with the present data are reports by several other investigators of a reduction in both renal and testicular lipid peroxidation in animals treated with dithiocarbamates following challenge with a variety of heavy metals (47, 48).

Clinical trials of the efficacy of NAC in patients with ARDS have demonstrated either a complete absence of efficacy (49) or a modest effect on outcome (50). The present study examined the mechanisms by which PDTC, a far more potent antioxidant, modulated acute lung injury in an animal model of ARDS. Data presented herein demonstrate that PDTC effectively mitigates LPS-induced acute lung injury. This effect is not mediated through the inhibition of gene activation, but through the potent antioxidant effects of PDTC. The use of dithiocarbamates in cases of heavy-metal poisoning and latent HIV infection (21, 22) provides evidence of the safety of these agents in humans. Thus, PDTC may represent an agent with therapeutic potential in ARDS and other disease states characterized by neutrophil-mediated oxidative tissue injury.