Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The development of acute lung injury significantly contributes to morbidity in patients after major trauma (1, 2). Recent studies suggest that the global ischemia/reperfusion related to resuscitation from hemorrhagic shock renders the trauma victim primed for an exaggerated response to a second, often trivial, inflammatory stimulus, the so-called "two-hit" hypothesis (3). For example, neutrophils recovered from trauma patients release increased amounts of superoxide anion, proteases, and proinflammatory cytokine in response to formyl-methionyl-leucyl-phenylalanine when compared with normal controls (4, 5). Presumably, sequestration of these activated cells in various organs predisposes to tissue injury and the onset of organ dysfunction (6). The ability of a second hit to cause organ injury after resuscitated shock has also been demonstrated in the experimental setting. For example, using a rodent model of shock/resuscitation, we have reported that antecedent shock in rodents primes for increased lung injury in response to a subsequent small dose of endotoxin (7). In addition to neutrophils, priming of other cell populations including alveolar macrophages and circulating macrophages as well as endothelial cells appears to occur and likely contributes to enhanced lung injury and the concomitant alveolar neutrophil sequestration (7).

Fibrin deposition within the air space is one of the hallmarks of the acute respiratory distress syndrome (ARDS) (10). Alveolar fibrin deposits appear to contribute to the magnitude of the inflammatory response by virtue of the ability of their cleavage and degradation products to promote chemotaxis, to increase vascular permeability, and to exert modulatory effects on various immune cells (11). In addition, fibrin may participate in the resolution phase of ARDS, possibly contributing to lung fibrosis by providing a matrix for macrophage migration and by promoting angiogenesis and collagen deposition (15).

The degree of alveolar fibrin deposition represents a net balance between procoagulant and anticoagulant and/or fibrinolytic activities in the lung. Previous studies have demonstrated that local abnormalities in these parameters, characterized by increased procoagulant activity (PCA) and reduced fibrinolysis, predispose to aberrant fibrin deposition in the alveolar space in humans as well as in experimental models of acute lung injury (18). For example, Idell and colleagues reported that the bronchoalveolar lavage (BAL) fluid (BALF) from patients with ARDS had increased tissue factor (TF)-Factor VII-dependent PCA and elevated plasminogen activator inhibitor (PAI) levels, while fibrinolytic activity was found to be reduced (18, 19). Interestingly, patients considered at risk for the development of ARDS by virtue of having sustained major trauma exhibited a small rise in PCA compared with normal control subjects, but not to the extent observed in patients with ARDS (23). This suggests the possibility that trauma may have primed the lungs for enhanced fibrin deposition in those proceeding to profound ARDS.

In the present studies, a two-hit model of resuscitated hemorrhagic shock followed by intratracheal lipopolysaccharide (LPS) in the rodent was used to evaluate the cellular mechanisms underlying the enhanced fibrin deposition in the alveolar space. The data demonstrate that, although shock alone has little effect on the procoagulant milieu of the lung, it primes the lung for increased macrophage tissue factor-dependent PCA activity and enhanced PAI in response to a small dose of LPS. Further, the presence of tumor necrosis factor (TNF) in the BALF appears to be required for this heightened net procoagulant response, although it does not contribute to chemokine-induced alveolar neutrophilia. Thus, after resuscitated shock, both TNF-dependent and -independent pathways are involved in mediating the development of lung fibrin deposition and neutrophil sequestration, the pathologic hallmarks of ARDS.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animal Model of Hemorrhage/Resuscitation

A model of hemorrhage/resuscitation in rodents was used as described elsewhere (7). Male Sprague-Dawley rats (300 to 350 g; Charles River, St. Constant, PQ, Canada) were anesthetized with 80 mg/kg ketamine and 8 mg/kg xylazine administered intraperitoneally. The right carotid artery was cannulated with a 22-gauge angiocath (Becton-Dickinson, Franklin Lakes, NJ) for monitoring of mean arterial pressure (MAP), blood sampling, and resuscitation. Hemorrhagic shock was initiated by blood withdrawal and reduction of the MAP to 40 mm Hg within 15 min. This blood pressure was maintained by further blood withdrawal if the MAP was > 45 mm Hg, and by infusion of 0.5 ml Ringer's lactate (RL) if the MAP was < 35 mm Hg. Shed blood was collected into 0.1 ml citrate/ml blood to prevent clotting. After a hypotensive period of 60 min, animals were resuscitated by transfusion of the shed blood and RL in a volume equal to that of shed blood over a period of 2 h. In some studies, animals received N-acetyl-cysteine (NAC) (0.5 g/kg) via the artery before the infusion of RL. The catheter was then removed, the carotid artery ligated, and the cervical incision closed. Sham animals underwent the same surgical procedures, but hemorrhage was not induced. NAC delivery in sham animals was performed at an equivalent time to that in shock animals.

Two protocols were used to study cellular activation and lung injury after shock/resuscitation (Figure 1). The first protocol was performed in vivo. In this protocol, a tracheotomy with a 14-gauge catheter was performed midway through the 2-h resuscitation period. At the end of resuscitation, either LPS (Escherichia coli; O111:B4, 30 µg/kg in 200 µl saline) or saline (SAL) alone was administered intratracheally followed by 20 mechanically ventilated breaths using a rodent ventilator. The animals were therefore assigned to one of four groups: sham/SAL, shock/SAL, sham/LPS, or shock/LPS. The second protocol consisted of recovering alveolar macrophages by BAL at the end of shock/resuscitation and then culturing them in vitro for subsequent study. Briefly, at the end of the resuscitation period, BAL was performed on shocked or sham animals and macrophages were isolated as described later. At this time, the alveolar cell content did not differ between sham and shock animals. The macrophages obtained from hemorrhage/resuscitated or sham rats were then incubated for 1, 2, 4, and 6 h at 37°C in 5% CO₂ either alone or in the presence of 0.1 µg/ml LPS. At the end of the incubation period, cells were pelleted by centrifugation at 300 × g for 10 min. In some studies, supernatants and cells resuspended at 10⁶ cells/ ml RPMI 1640 were frozen at 70°C for later measurement of TNF and PCA, respectively, whereas in other studies, cells were used for RNA extraction for Northern blot analysis.

View larger version (13K): [in this window] [in a new window]   Figure 1.   A schematic diagram showing the experimental protocol used in the present studies. At the end of the resuscitation period, animals either were treated in vivo with intratracheal LPS and then killed at various time points for recovery of lung tissue or, alternatively, underwent BAL for recovery of alveolar macrophages for subsequent culture in vitro with or without addition of LPS.

For the in vivo anti-TNF antibody blockade experiment, rats were given an intratracheal instillation of 100 µl rabbit antimouse TNF- neutralizing antiserum, known to also neutralize rat TNF- (Genzyme Diagnostics, Cambridge, MA) or rabbit nonimmune immunoglobulin (Ig)G in 100 µl SAL 10 min before the intratracheal LPS instillation. Animals were killed at various time points by pentobarbitol overdose.

Assessment of Lung Injury

To determine whether in vivo manipulation caused disruption of the alveolar-capillary barrier and leakage of albumin into the alveolar space, alveolar albumin accumulation was assessed by injecting 2 µCi [¹²⁵I]albumin in a total volume of 0.2 ml SAL into the tail vein immediately after intratracheal LPS or SAL (24). At the end of the experimental protocol, 1 ml of blood was withdrawn by cardiac puncture for determining counts per minute (cpm). After exsanguination, lungs were perfused via a cannula in situ with 10 ml phosphate-buffered saline (PBS). The perfused PBS was withdrawn gently and aliquotted into 1 ml/tube for counting cpm. The alveolar albumin accumulation was normalized to blood cpm as follows: alveolar albumin accumulation = BALF cpm/ml divided by blood cpm/ml.

BAL

BAL was performed to recover cells from the alveolar space. The lungs were lavaged via the intratracheal angiocath with cold PBS, 8 mM sodium phosphate, 2 mM potassium phosphate, 0.14 M sodium chloride, and 0.01 M potassium chloride, pH 7.4, with 0.1 mM ethylenediaminetetraacetic acid (EDTA). A volume of 40 ml PBS was collected from each rat and centrifuged at 300 × g for 10 min (24). To assess cell number, supernatant was discarded and the pelleted cells were resuspended in a small volume of serum-free Dulbecco's modified Eagle's medium (DMEM) culture medium (GIBCO BRL, Burlington, ON, Canada). Total cell counts were determined on a grid hemocytometer. Differential cell counts were enumerated on cytospin-prepared slides that were stained with Wright-Giemsa stain. A total of 500 cells were counted in cross-section per sample and the numbers of neutrophils and alveolar macrophages were calculated as the total cell count times the percentage of the respective cell type in the BALF sample. In studies of macrophage function, the cell pellet was suspended in Neutrophil Isolation Medium (Cardinal Associates, Inc., Santa Fe, NM) and centrifuged at 750 × g, 20°C for 45 min for macrophage isolation (25). The isolated macrophages were washed in 5 ml of modified (calcium- and magnesium-free) Hanks' balanced salt solution and centrifuged again at 300 × g for 10 min. The pellet was resuspended in DMEM culture medium containing 10% fetal calf serum at a concentration of 1 × 10⁶ cells/ml medium and aliquotted into polypropylene tissue culture tubes. This technique generated a cell suspension with a viability in excess of 95% as assessed by trypan blue exclusion, and a cell population of > 95% macrophages as assessed by Wright- Giemsa staining.

Measurement of PCA and TNF

Macrophages were disrupted by freeze-thawing and PCA was determined using the single-stage recalcification clotting assay (26). PCA was expressed as units per 10⁶ cells by comparison with rabbit-brain thromboplastin. In a typical experiment, macrophages recovered from shock animals and then treated with LPS in vitro for 4 h shortened clotting times from 70 to 41 s, representing an increase in PCA from 1.8 U/10⁶ cells to 25 U/10⁶ cells. TNF in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA), as previously described (27).

Northern Blot Analysis

Total RNA from whole-lung tissue or alveolar macrophages was obtained using the guanidium-isothiocyanate method (28). Briefly, lungs were harvested from treated animals and immediately frozen in liquid nitrogen. Macrophages were recovered by BAL from treated rats. Lungs or macrophages were then thawed and homogenized in 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 nylon membrane. Hybridization was carried out using a [³²P]deoxycytidine triphosphate- labeled TF complementary DNA (cDNA) and a [³²P]adenosine triphosphate (ATP)-end-labeled 30-base oligonucleotide probe for cytokine-induced neutrophil chemoattractant (CINC), which is complementary to nucleotides 134 to 164 of CINC cDNA (see Reference 29; kindly provided by Dr. Timothy S. Blackwell, Vanderbilt University School of Medicine, Nashville, TN). The cDNA probes for plasminogen activator (PA) and PAI-1 were obtained from ATCC (Rockville, MD). Blots were then washed under conditions of high stringency and specific messenger RNA (mRNA) bands were detected by autoradiography in the presence of intensifying screens as reported elsewhere (24). Blots were stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase (G3PDH), which is a ubiquitously expressed housekeeping gene to control for loading (30). Expression of mRNA was quantitated using a PhosphorImager and accompanying ImageQuant software (Molecular Dynamics, Sunnyvale, CA), and was normalized to the G3PDH signal.

Nuclear Protein Extraction

Nuclear protein was extracted from the macrophages or from lung tissue by the method of Deryckere and Gannon (31) and nuclear factor (NF)-B translocation was measured by electrophoretic mobility shift assay (EMSA) (32). For lung tissue, aliquots of 200 to 500 mg of frozen tissue were ground to powder with a mortar in liquid nitrogen. The thawed powder was homogenized in a Dounce tissue homogenizer with 4 ml of solution A (0.6% Nonidet P-40 [NP-40], 150 mM NaCl, 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes] [pH 7.9], 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]). The cells were lysed with five strokes of the pestle. After transfer to a 15-ml tube, debris was pelleted by briefly centrifuging at 2,000 rpm for 30 s. The supernatant was transferred to 50-ml Corex tubes, incubated on ice for 5 min, and centrifuged for 10 min at 5,000 rpm. Nuclear pellets were then resuspended in 300 µl of solution B (25% glycerol, 20 mM Hepes [pH 7.9], 420 mM NaCl, 1.2 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol [DTT], 0.5 mM PMSF, 2 mM benzamidine, 5 µg/ml pepstatin, 5 µg/ml leupeptin, and 5 µg/ml aprotinin) and incubated on ice for 20 min. The mixture was transferred to microcentrifuge tubes, and nuclei were pelleted by centrifugation at 14,000 rpm for 1 min. Supernatants containing nuclear proteins were aliquotted in small fractions, frozen in liquid nitrogen and stored at 70°C. Protein quantitation was performed using the Bio-Rad protein assay dye reagent (Bio-Rad, Hercules, CA).

EMSA

Oligonucleotides used for protein binding were: to the TF B-like site, 5'-GTCCCGGAGTTTCCTACCGGG-3' (33); and to the TNF B₃ binding site, 5'- CAAACAGGGGGCTTTCCCTCCTC-3' (34). End labeling was performed by T4 kinase in the presence of [³²P]ATP. Labeled oligonucleotides were purified on a Sephadex G-50 M column (Pharmacia Biotech, Inc., Piscataway, NJ). An aliquot of 5 µg of nuclear protein was incubated with the labeled double-stranded probe (~ 50,000 cpm) in the presence of 5 µg of nonspecific blocker, poly(dI-dC) in binding buffer (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.2% NP-40, and 0.5 mM DTT) at 25°C for 20 min. Specific competition was performed by adding 100 ng of unlabeled double-stranded TF oligonucleotide; for nonspecific competition, 100 ng of unlabeled double-stranded mutant TF oligonucleotide 5'-GTCCCGGAGTTAGATACCGGG-3' that does not bind NF-B was added. In studies of TNF, the unlabeled double-stranded mutant TNF probe was 5'-CAAACAGGCTTTCCCTCCTC-3'. The mixture was separated by electrophoresis on a 5% polyacrylamide gel in 1× Tris glycine EDTA buffer. Gels were vacuum-dried and subjected to autoradiography and PhosphorImager analysis.

Measurement of Neutrophil Chemiluminescence

Rat neutrophils were isolated from BALF by centrifugation (750 × g, 20°C for 45 min) in NIM.2 neutrophil isolation medium (Cardinal Associates). The isolated neutrophils were washed with 5 ml of DMEM and suspended in DMEM in 1 × 10⁶ cells/ml concentration. To 1 ml of the cell sample, 10 µl of 100 µM dihydrorhodamine 123 (Molecular Probes, Eugene, OR) was added, and the mixture was incubated at 37°C for 5 min. Chemiluminescence was measured by the FACScan (Becton-Dickinson, Palo Alto, CA) using an FL1 detector (35). Typically, 5,000 cells were analyzed per condition. For comparison, the starting control value for each animal was normalized to 100 and subsequent readings were compared relative to this.

Statistics

The data are presented as means ± standard error of the mean (SEM) of n determinations as indicated in the figure legends. Data were analyzed by one-way analysis of variance; post hoc testing was performed using the Bonferroni modification of the t test. Individual studies shown are representative of at least three independent experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Resuscitated Shock Predisposes for Increased Lung Fibrin Deposition

To determine the ability of shock to promote lung fibrin deposition, TF expression in the lung was evaluated. As shown in Figure 2, LPS alone caused a small rise in TF mRNA levels, whereas shock alone had little effect. However, the combination of shock plus LPS caused a marked further rise in TF mRNA expression. To evaluate the biologic activity of this increase, the PCA of whole lung was studied using the one-stage recalcification assay (Figure 3). Shock alone did not induce PCA, whereas the low dose of LPS administered intratracheally caused a small rise. By contrast, this dose of LPS after resuscitation from shock caused a ~ 6-fold increase compared with LPS alone. Failure to clot Factor VII-deficient plasma suggests that the increased PCA was predominantly due to augmented TF expression (data not shown).

View larger version (57K): [in this window] [in a new window]   Figure 2.   Effect of shock on LPS-induced TF mRNA expression in the lung. Northern blot analysis was performed on total RNA extracted from the whole lung tissue of shocked or sham rats at t = 4 h after intratracheal LPS or SAL. Corresponding G3PDH mRNA bands are shown as evidence of comparable loading. The blot is representative of four independent studies.

View larger version (15K): [in this window] [in a new window]   Figure 3.   Whole-lung PCA in sham or shock animals 4 h after treatment with intratracheal LPS or SAL. PCA was assessed using the single-stage recalcification clotting assay as described in MATERIALS AND METHODS. The data represent the means ± SEM of studies performed in four rats in each group (*P < 0.01 versus other groups).

Several cell types in the lung have been identified as possible sources of TF, including alveolar macrophages, alveolar epithelial cells, and endothelial cells (36). To evaluate the contribution of alveolar macrophages, these cells were recovered and enriched after resuscitated shock (or sham treatment) and then treated in vitro with or without LPS for 4 h. Figure 4A shows that macrophages recovered from animals that were shocked and then exposed to LPS exhibited a marked enhancement in TF mRNA expression compared with cells from shocked animals alone or those from sham animals treated with LPS in vitro. As observed in whole-lung samples, the enhanced TF mRNA expression correlated well with the PCA in these cells (Figure 4B). Considered together, these studies demonstrate that antecedent shock primes the lung for augmented PCA and that a significant component of this effect is due to increased macrophage TF expression.

View larger version (27K): [in this window] [in a new window]   Figure 4.   (A) Northern blot analysis and scanning densitometry showing the effect of shock/LPS on TF mRNA expression in alveolar macrophages. Alveolar macrophages were recovered and enriched after resuscitated shock or sham treatment and then treated in vitro with or without LPS (0.1 µg/ml) for 4 h. Total RNA was extracted from the cells, and Northern blot analysis was performed. Scanning densitometry values of Northern blot for TF mRNA are normalized by densitometry of corresponding G3PDH mRNA bands and expressed as means ± SEM (n = 4 rats per group; *P < 0.01 versus other groups). (B) Effect of shock on LPS-induced PCA in alveolar macrophages. Alveolar macrophages were obtained using the same experimental conditions as described in Figure 3A. Values are means ± SEM (n = 4 for each group; *P < 0.05 versus sham/SAL and shock/SAL; **P < 0.01 versus sham/LPS).

The promoter region of the TF gene contains an NF-B consensus binding sequence, which has previously been shown to be necessary for activation of the TF gene in response to proinflammatory stimuli (39). Figure 5 shows a time course of NF-B translocation and binding to the TF gene-specific NF-B consensus binding sequence using isolated alveolar macrophages from sham or shock animals treated for various amounts of time with LPS. NF-B translocation occurred earlier and to a greater extent in response to LPS treatment in animals exposed to prior resuscitated shock compared with sham animals. Cold probe competed for binding whereas mutant probe did not, indicating the specificity of the reaction. Cells derived from sham or shock animals did not show NF-B binding when incubated over this time period in the absence of LPS (data not shown).

View larger version (87K): [in this window] [in a new window]   Figure 5.   Time course for shock/LPS-induced NF-B nuclear translocation in alveolar macrophages. Alveolar macrophages were isolated from sham or shock animals and treated for the indicated times with LPS. The probe for the EMSA was a [32P]ATP end-labeled, double-stranded construct corresponding to the sequence of the TF-B-like site. Cold competition (cold) and mutant nonspecific competition (NS) are also shown for the shock/ LPS sample at t = 4 h. A representative autoradiograph of three independent experiments is shown.

We also investigated whether shock might modulate components of the fibrinolytic pathway, thereby contributing to net fibrin deposition in the lung. Figure 6A shows the levels of PA mRNA in the lung under various conditions. PA mRNA was constitutively expressed after sham manipulation and was slightly increased by shock alone. Administration of LPS caused ~ 40% reduction in these levels, an effect which was not modulated by antecedent shock. By contrast, levels of PAI-1 mRNA, one of the major antifibrinolytics in the lung in ARDS (19), were clearly increased by LPS, an effect that was magnified ~ 2-fold by antecedent shock (Figure 6B). Neither sham nor shock alone showed evidence of PAI-1 mRNA expression. Thus, when considered in aggregate with the TF studies, these findings suggest that shock primes the lung for enhanced fibrin deposition by increasing the PCA as well as inhibiting fibrinolysis.

View larger version (39K): [in this window] [in a new window]   Figure 6.   (A) Northern blot analysis and scanning densitometry showing the changes in PA mRNA level in the lung in sham or shock animals with or without LPS treatment. Total RNA was extracted from the lung tissue from shock or sham animals at 4 h after intratracheal LPS or SAL. Scanning densitometry values for Northern blot for PA mRNA are normalized by densitometry of corresponding G3PDH mRNA bands and expressed as means ± SEM (n = 3 rats per group; *P < 0.05 versus sham/SAL and shock/SAL). (B) Representative Northern blot analysis showing the effect of shock/LPS on PAI-1 mRNA expression in the lung. Northern blot was performed on the same samples of total RNA as described in Figure 5A. Scanning densitometry values are means ± SEM (n = 3 rats per group; *P < 0.05 versus sham/SAL and shock/ SAL; **P < 0.01 versus sham/LPS).

Role of TNF in Modulating Lung PCA

Because TNF has been shown in vitro to promote TF expression in monocytes/macrophages (40), we hypothesized that TNF might play a role in modulating TF expression in vivo. Macrophages were recovered from animals after resuscitated shock or sham treatment and then exposure to LPS in vitro. As shown for TF expression, shock also primed for enhanced TNF expression (Figure 7). Specifically, although LPS alone caused a rise in TNF compared with shock or sham alone, cells treated with LPS after antecedent shock released significantly more TNF into the supernatant than did sham cells exposed to LPS at all time points (Figure 7A). This enhancement was also observed at the level of TNF gene expression, as evidenced by the rise in levels of TNF mRNA in the shock/LPS group compared with all other groups (Figure 7B).

View larger version (35K): [in this window] [in a new window]   Figure 7.   (A) TNF- concentrations in macrophage supernatant. Alveolar macrophages were recovered from animals after resuscitated shock or sham treatment and then incubated with or without LPS (0.1 µg/ml) for the time indicated. TNF- levels in the supernatant were detected with ELISA as described in MATERIALS AND METHODS. Values are expressed as means ± SEM (n = 6; *P < 0.05 versus sham/SAL, shock/SAL, and sham/LPS at same time point; **P < 0.01 versus sham/SAL, shock/SAL, and sham/LPS at same time point). (B) Northern blot showing the effect of shock on LPS-induced TNF- mRNA expression in alveolar macrophages at t = 4 h. Total RNA was extracted from the alveolar macrophages, which were isolated and treated as described in Figure 6A. G3PDH mRNA was used to ensure comparable loading between lanes. The blot is representative of three independent studies.

To determine whether TNF contributed to the induction of macrophage TF expression in vivo, anti-TNF antibody was administered intratracheally just before LPS instillation. As shown in Figure 8A, anti-TNF antibody reduced levels of TF mRNA in shock/LPS animals to that seen in sham/LPS animals, suggesting a role for TNF in the priming for TF expression in shock/LPS animals. Anti-TNF also lowered TF mRNA levels in sham/LPS animals as well as in shock-alone animals, albeit not to control levels. Similar results were noted when biologic PCA was assessed (Figure 8B). Anti-TNF antibody completely reversed the priming for increased LPS-induced PCA seen in the animals exposed to antecedent shock. In sham/LPS animals, TNF blockade caused only a slight reduction in PCA. Figure 8C shows that TNF blockade also inhibited NF-B translocation in shock/LPS animals, consistent with its role in TF mRNA expression. These findings therefore suggest that TNF plays an important role in the shock-induced priming of the lung for increased TF after LPS. This appears to be related to its ability to promote NF-B translocation and activation in alveolar macrophages, although further study using specific inhibition of NF-B is required.

View larger version (42K): [in this window] [in a new window]   Figure 8.   (A) Effect of anti-TNF- antibody on TF mRNA expression. Rats were given an intratracheal instillation of 100 µl of rabbit antimouse TNF- neutralizing antiserum (Ab) or rabbit nonimmune IgG (IgG) in 100 µl of SAL at 50 min after resuscitated shock or sham operation and 10 min before the intratracheal LPS or SAL instillation. At 4 h after LPS or SAL administration, lung tissue was collected for Northern blot analysis. The figure is representative of three studies. (B) Whole-lung PCA after anti-TNF- antibody treatment (t = 4 h). Animals were treated as in Figure 7A with lung tissue recovered from t = 4 h for PCA determination. Values are means ± SEM (n = 4 rats per group; *P < 0.01 versus sham/SAL, shock/SAL, and sham/LPS; **P < 0.01 versus shock/LPS with IgG). B and C: Ab, anti-TNF antibody; IgG, nonimmune IgG. (C) Representative EMSA showing the effect of anti-TNF- antibody on NF-B translocation in the lung. Animals were treated as in Figure 7A but lung tissue was recovered at t = 2 h for EMSA. The figure is representative of three studies.

The role of TNF in alterations in PA and PAI-1 expression was also studied. Anti-TNF treatment prevented the LPS-induced rise in PAI-1 mRNA levels and also lessened, but did not abolish, the exaggerated response observed in shock/LPS animals (Figure 9A). Further, anti-TNF caused partial reversal of the reduction in PA expression in both the sham/LPS and shock/LPS animals (Figure 9B).

View larger version (72K): [in this window] [in a new window]   Figure 9.   (A) Effect of anti-TNF- antibody on PAI-1 mRNA levels in the lung. Anti-TNF- antibody or rabbit nonimmune IgG was given intratracheally 50 min after resuscitated shock or sham operation, and 10 min before LPS or SAL instillation. The lung was sampled at 4 h after LPS or SAL treatment. A Northern blot shows the alterations of PAI-1 mRNA expression in the lung after anti-TNF- antibody treatment. A representative of three similar studies is illustrated. (B) Effect of anti-TNF- antibody on PA mRNA levels in the lung. Anti-TNF- antibody or rabbit nonimmune IgG was given intratracheally 50 min after resuscitated shock or sham operation, and 10 min before LPS or SAL instillation. The lung was sampled at 4 h after LPS or SAL treatment. A Northern blot shows the alterations of PA mRNA expression in the lung after anti-TNF- antibody treatment. A representative of three similar studies is illustrated.

Effect of Anti-TNF Treatment on Neutrophil Sequestration in the Lung

Our previous studies demonstrated that antecedent shock primed for increased lung injury and neutrophil sequestration by causing an early and augmented rise in the expression of the chemokine CINC (7). The effect of TNF blockade on alveolar neutrophilia was studied. As previously reported, shock/LPS caused a marked increase in BALF neutrophil counts compared with LPS and with shock alone (Figure 10A). However, anti-TNF antibody had no effect on this enhancement of alveolar neutrophil accumulation. Consistent with this finding, anti-TNF treatment had no effect on the shock-induced priming for increased CINC mRNA levels after LPS treatment (Figure 10B).

View larger version (44K): [in this window] [in a new window]   Figure 10.   (A) Effect of anti-TNF- antibody on neutrophil counts in BALF from shocked and sham rats at t = 4 h after treatment with LPS or SAL. Neutrophil recovery was performed as described in MATERIALS AND METHODS (*P < 0.05 versus sham/ SAL and shock/SAL; **P < 0.01 versus sham/SAL, shock/SAL, and sham/LPS). (B) Effect of anti-TNF antibody on levels of whole-lung CINC mRNA from shocked and sham rats at t = 4 h after treatment with LPS or SAL. A representative of three separate studies is illustrated.

Permeability of [¹³¹I]albumin into the lung after anti-TNF treatment was examined as a measure of lung injury. As shown in Figure 11A, antecedent shock augmented albumin leakage after LPS compared with sham animals treated with LPS. TNF blockade totally prevented this rise. To discern whether TNF contributed to neutrophil activation as a mechanism responsible for its protective effect, we examined production of reactive oxygen species of alveolar neutrophils using the chemiluminescence response. As shown in Figure 11B, neutrophils recovered from the BALF of shock/ LPS animals exhibited marked chemiluminescence compared with control or sham/LPS animals. However, prior treatment with anti-TNF antibody prevented this increased response. In sham and shock-alone animals, we were unable to recover sufficient numbers of alveolar neutrophils to measure their chemiluminescence. However, as shown in Figure 11B, neutrophils recovered from the blood of shock animals exhibited a rise in chemiluminescence compared with sham animals. Considered together with the neutrophil data presented earlier, these findings suggest that, although TNF plays a minor role in the regulation of neutrophil sequestration after shock priming, it contributes significantly to the activation of these cells and resultant lung injury.

View larger version (28K): [in this window] [in a new window]   Figure 11.   (A) Effect of anti-TNF- antibody on transpulmonary albumin flux. Rats were given an intratracheal instillation of 100 µl of rabbit antimouse TNF- neutralizing antiserum or rabbit nonimmune IgG in 100 µl of SAL 50 min after resuscitated shock or sham operation and 10 min before the intratracheal LPS or SAL instillation. Transpulmonary albumin flux was measured as described in MATERIALS AND METHODS. The blood and BALF were collected at 6 h after LPS or SAL treatment. Values are means ± SEM (n = 3 rats per group; *P < 0.05 versus sham/SAL, shock/SAL, and sham/LPS; **P < 0.05 versus shock/LPS, no-treatment, and IgG groups). (B) Effect of anti-TNF- antibody on neutrophil chemiluminescence. Animals were treated as described in Figure 10A. BALF was collected at 4 h after LPS or SAL instillation. Neutrophil chemiluminescence was measured by flow cytometry as described in MATERIALS AND METHODS. Values are expressed in mean channel fluorescence (MCF). Data represent means ± SEM for three animals per group. (*P < 0.05 versus sham/SAL and sham/LPS; **P < 0.05 versus shock/LPS IgG.)

Effect of N-AC on TNF and TF Gene Expression in the Lung

We and others have shown that antioxidants are able to modulate CINC expression in the lung (7, 29). To determine their effect on TNF and TF expression, NAC was included in the resuscitation fluid as part of the shock/resuscitation protocol. Figure 12A shows that shock-induced priming for increased TNF expression was blocked by use of NAC, as was TF expression, consistent with its position downstream of TNF. By contrast, NAC had little effect on the TNF mRNA levels induced by LPS alone. The effect of NAC on TNF-gene specific NF-B translocation in the lung was also evaluated. As shown in Figure 12B, NAC prevented the augmented NF-B translocation observed in the shock/LPS animals, but had little effect on NF-B translocation induced by LPS alone.

View larger version (65K): [in this window] [in a new window]   Figure 12.   (A) Northern blot showing the effect of NAC (0.5 g/kg) on expression of TNF- and TF mRNA extracted from the lung tissue of shock and sham animals 4 h after intratracheal LPS instillation. A representative of three independent experiments is shown. (B) EMSA demonstrating the effect of NAC on NF-B nuclear translocation. Lung tissue from shock or sham animals was sampled 2 h after LPS or SAL treatment. EMSA was performed with a [32P]ATP end-labeled construct corresponding to the sequence of TNF- B enhancer 3. Cold competition (cold) and mutant nonspecific competition (NS) are also shown for the shock/LPS sample. A representative of three independent experiments is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Fibrin deposition is one of the histopathologic hallmarks of acute lung injury and is considered to contribute to the pathogenesis of injury and its subsequent resolution (10). The present studies demonstrate that global ischemia/reperfusion initiated by resuscitated hemorrhagic shock primes the lungs for a net procoagulant response after a second inflammatory stimulus, i.e., low-dose intratracheal endotoxin. This response is characterized by increased levels of tissue factor and PAI mRNA as well as a reduction in PA mRNA. The biologic relevance of the elevated TF mRNA is indicated by the demonstration of increased Factor VII- dependent PCA in whole-lung homogenates, a finding consistent with increased TF expression. These findings were recapitulated in alveolar macrophages recovered after resuscitated shock, suggesting that these cells were significant contributors to the effects observed. Although these cells were enriched following their recovery, we cannot rule out the possibility that other cell types, particularly epithelial and endothelial cells, might have been minor contaminants in the cell preparation or that they might have contributed to the observations made on whole lung homogenates, inasmuch as both cell types are known to express procoagulant and fibrinolytic molecules (36, 37, 41, 42). In addition, shock/LPS also markedly enhanced production of TNF mRNA and protein in the lung compared with LPS alone. The finding that intratracheal anti-TNF antibody reversed the enhanced expression of TF and PAI mRNAs and also prevented the reduction in PA mRNA suggests that TNF plays an important upstream role in the augmented fibrin deposition observed in the lung during acute injury. From a functional standpoint, this conclusion is supported by the finding that anti-TNF prevents the shock-induced priming for increased whole-lung PCA. These data thus provide in vivo correlation for previous in vitro studies showing that TNF can contribute to regulation of TF (40), and also support prior reports showing that PAI expression is modulated, in part, by TNF release after LPS administration in vivo (43, 44).

Our previous studies reported that the augmented release of the C-X-C chemokine CINC by alveolar macrophages was responsible for the increased alveolar neutrophil counts after shock/LPS treatment compared with sham/ LPS animals (7). In contrast to its effect on TF and PAI, anti-TNF antibody had no effect on the expression of CINC mRNA. This lack of inhibition was also demonstrated by the finding that BALF neutrophil numbers did not differ between animals treated with and without anti-TNF antibody. These findings thus suggest separate signaling pathways for shock-induced priming for lung fibrin deposition and neutrophil infiltration in this model, one TNF-dependent and the other TNF-independent. However, the data clearly suggest that TNF is not without effect on the neutrophil arm of these pathways. While having no effect on neutrophil sequestration, TNF inhibition reduced lung injury in this model, as measured by permeability to [¹³¹I]albumin. Because in vitro studies had previously demonstrated that TNF is able to prime neutrophils for increased release of reactive oxygen species (45, 46), we postulated that the protection seen after anti-TNF treatment may have been related to the reduced level of neutrophil activation. Consistent with this hypothesis, our studies showed that the chemiluminescence exhibited by BALF neutrophils from animals treated with anti-TNF was significantly reduced compared with those given nonspecific Ig.

The discrete role of TNF as a contributor to neutrophil activation and lung injury, but not to neutrophil sequestration in the lung, has been reported by other investigators. Hybertson and associates demonstrated that intravenous administration of TNF binding protein with concomitant intratracheal interleukin-1 resulted in pathophysiologic changes reported in the present study using anti-TNF antibody (47). Specifically, TNF binding protein inhibited lung injury, but was without effect on neutrophil accumulation or CINC levels. Studies by Ulich and colleagues similarly demonstrated a minor role for TNF in the early neutrophil sequestration after intratracheal LPS (48, 49). During the first 4 h after LPS, TNF antagonism with recombinant soluble human TNF receptor type 1 had no effect on the magnitude of BAL neutrophilia in a rodent model (48). In addition, TNF at relatively high doses was shown to induce a rather modest degree of alveolar neutrophilic infiltrate (49). Considered in aggregate, these data suggest that the role of TNF with respect to neutrophils in lung injury is related more to its ability to activate these cells than to induce their extravascular accumulation.

Although CINC and TNF appear to influence separate downstream events in the pathogenesis of lung injury after resuscitated shock, their upstream regulation in this model appears to be similar. Specifically, the augmented expression of TNF after shock/LPS appears to involve increased nuclear translocation of the important transcription factor NF-B. Further, the addition of the antioxidant NAC to the resuscitation fluid prevented the priming for increased expression of TNF mRNA after LPS. NAC also abrogated the augmented TF mRNA levels, presumably related to its effect on TNF. The ability of NAC supplementation to prevent priming in this model is similar to that previously reported for CINC expression and implies that oxidant stress generated during hemorrhage/resuscitation is a significant contributor to the induction of the primed state. Studies by other investigators suggest that xanthine oxidase produced after ischemia/reperfusion may be primarily responsible for elaboration of these oxidants (50). A recent report demonstrated that upregulation of inducible nitric oxide synthase during the shock phase of shock/ resuscitation may be essential to the development of lung injury, a finding consistent with a conclusion that cellular events occurring during the hypoxic phase may also contribute to the primed state (53).

The present studies demonstrate that shock/resuscitation induces a state in the lung wherein its cellular constituents are primed for expressing procoagulant and antifibrinolytic molecules in response to an inflammatory stimulus, while simultaneously reducing its fibrinolytic capacity. The upstream role of TNF and its regulation by oxidant stress suggest targets for therapeutic or preventive intervention during the resuscitation phase of patient management.