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Am J Physiol Lung Cell Mol Physiol 287: L1248-L1255, 2004. First published August 6, 2004; doi:10.1152/ajplung.00164.2004
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Therapeutic effect of in vivo transfection of transcription factor decoy to NF-{kappa}B on septic lung in mice

Naoyuki Matsuda,1 Yuichi Hattori,2 Yoshika Takahashi,1 Jun Nishihira,3 Subrina Jesmin,1 Masanobu Kobayashi,4 and Satoshi Gando1

1Department of Anesthesiology and Critical Care Medicine, 2Department of Pharmacology, and 3Department of Biochemistry, Hokkaido University Graduate School of Medicine, and 4Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-8638, Japan

Submitted 6 May 2004 ; accepted in final form 27 July 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nuclear factor-{kappa}B (NF-{kappa}B) plays a key role in regulating expression of several genes involved in the pathophysiology of endotoxic shock. We investigated whether in vivo introduction of synthetic double-stranded DNA with high affinity for the NF-{kappa}B binding site could block expression of genes mediating pulmonary vascular permeation and thereby provide effective therapy for septic lung failure. Endotoxic shock was induced by an intravenous injection of 10 mg/kg Escherichia coli endotoxin in mice. We introduced NF-{kappa}B decoy oligodeoxynucleotide (ODN) in vivo 1 h after endotoxic shock by using a gene transfer kit. At 10 h, blood samples were collected for measurement of histamine and for blood-gas analysis. Gene and protein expression levels of target molecules were determined by means of Northern and Western blot analyses, respectively. The transpulmonary flux of 125I-labeled albumin was used as an index of lung vascular permeability. Administration of endotoxin caused marked increases in plasma histamine and gene and protein expressions of histidine decarboxylase, histamine H1 receptors, and inducible nitric oxide synthase in lung tissues. Elevated lung vascular permeability was also found. Blood-gas analysis showed concurrent decreases in arterial PO2, PCO2, and pH. All of these events induced by endotoxin were significantly inhibited by transfection of NF-{kappa}B decoy ODN but not by its mutated (scrambled) form (used as a control). Our results indicate for the first time the potential usefulness of NF-{kappa}B decoy ODN for gene therapy of endotoxic shock.

gene therapy; histamine; histidine decarboxylase; histamine H1 receptor; nitric oxide synthase; lung failure; endotoxic shock

SEPTIC SHOCK RESULTING FROM gram-negative bacterial infection is characterized by refractory hypotension, capillary leakage, and multiple organ dysfunction and is one of the most common reasons for death in intensive care units (28). Increased lung vascular permeability is thought to be a cause for lung failure with gram-negative sepsis (30). The marked lung vascular permeability witnessed in human clinical sepsis has been simulated in animal models by intravenous administration of Escherichia coli endotoxin (8). In later studies with animal models of sepsis (8), a number of potentially important chemical mediators in the pathogenesis of septic lung have been implicated.

Histamine is well known to increase pulmonary microvascular permeability (9). Endotoxemia in animals and patients elevates circulating histamine levels (13, 25, 31, 36). In our recent work using endotoxemic rabbits (20, 21), the sustained elevation of plasma histamine has been shown to be associated with the time-dependent increases in protein expression of histidine decarboxylase (HDC), by which histamine is synthesized from L-histidine in tissues (20, 21). Moreover, we have found that endotoxin administration causes superinduction of histamine H1 receptors in cardiovascular tissues of rabbits (20, 21). Earlier studies showed that blockade of histamine H1 receptors with diphenhydramine was effective in the increase in lung vascular permeability in animal models of sepsis (10, 34). Thus the hypothesis that histamine may, at least in part, mediate increased lung vascular permeability caused by endotoxemia is plausible.

Recent evidence suggests that many of the effects of septic mediators, including endotoxin, are mediated by increased production of nitric oxide (NO) (23). NO is synthesized from L-arginine by NO synthase (NOS). Although endothelial NOS (eNOS) is constitutively expressed and generates small amounts of NO in response to physical and receptor stimuli, inducible NOS (iNOS) produces much larger amounts of NO for sustained time periods and is principally implicated in the pathophysiological actions of NO. An important role for NO in septic shock has been suggested by observations that iNOS expression and increased NO production occurs during septic shock (37). NO has been reported to contribute to increased vascular permeation in response to inflammatory agonists, including endotoxin (6).

The mechanism by which the synthesis of multiple proteins contributed to inflammatory responses is triggered during gram-negative sepsis involves the activation of the transcriptional factor nuclear factor-{kappa}B (NF-{kappa}B). In unstimulated cells, NF-{kappa}B is present as a latent cytoplasmic complex bound to its inhibitor protein I-{kappa}B (2, 4). However, during sepsis, NF-{kappa}B can be activated by bacterial lipopolysaccharide (LPS) and inflammatory cytokines. Once activated, NF-{kappa}B dissociates from its inhibitor and translocates to the nucleus where it leads to the regulation of inflammatory molecule production (2, 4). The importance of the therapeutic strategy associated with prevention of NF-{kappa}B activation in septic shock has been emphasized in a few studies. Pyrrolidine dithiocarbamate, a potent inhibitor of NF-{kappa}B, has been shown to attenuate endotoxin-induced acute lung injury in rats (26). Furthermore, the anti-inflammatory agent parthenolide could protect against endotoxic shock in vivo by a specific inhibition of the NF-{kappa}B pathway (33).

Recently, Morishita et al. (24) demonstrated in rats that inhibition of NF-{kappa}B activation with the use of a decoy oligodeoxynucleotide (ODN) prevented myocardial infarction after coronary ligation. In the present study, we examined whether in vivo transfer of NF-{kappa}B decoy ODN could inhibit overexpression of the molecules involved in vascular leak and prevent increased lung vascular permeability in mice after LPS challenge. Thus we sought to evaluate the potential for improving lung failure after induction of sepsis with endotoxin when synthetic double-stranded DNA with high affinity for the NF-{kappa}B binding site was introduced.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care and housing. All animals received humane care in compliance with the guidelines of the Hokkaido University School of Medicine Animal Care and Use Committee (Sapporo, Japan). Male ICR mice weighing 25–30 g were quarantined in quiet, humidified, light-cycled rooms for 2–3 wk before use. Mice were allowed ad libitum access to food and water throughout quarantine.

Synthesis of ODN and selection of sequence targets. The following sequences of phosphorothioate double-stranded ODN against NF-{kappa}B binding site and of scrambled ODN were utilized in this study, which were the same as reported previously (24): 5'-CCTTGAAGGGATTTCCCTCC-3' and 3'-GGAACTTCCCTAAAGGGAGG-5' for NF-{kappa}B decoy ODN (consensus sequences are underlined); 5'-TTGCCGTACCTGACTTAGCC-3' and 3'-AACGGCATGGACTGAATCGG-5' for scrambled decoy ODN.

NF-{kappa}B decoy ODN has been shown to bind the NF-{kappa}B transcriptional factor (24). On the other hand, scrambled decoy ODN is a mutated form of wild-type NF-{kappa}B consensus sequence (27). Synthetic ODNs were initially dissolved in sterile Tris-EDTA buffer [10 mM Tris (pH 8.0), 1 mM EDTA], and the ODN concentration was quantitated by spectrophotometry. Then, after addition of 3 M CH3COONa and 99.8% ethanol, the mixture was centrifuged at 15,000 g, and the resulting pellet was washed in 70% ethanol. Ethanol was dried immediately before use.

Experimental protocol. Mice were intravenously injected with sterile saline or 10 mg/kg LPS (E. coli 055:B5; List Biological Laboratories, Campbell, CA) into the tail vein. For in vivo transfer of ODN, a gene transfer kit (In Vivo GeneSHUTTLE: GDS0090, Obiogene, Carlsbad, CA) was used. In Vivo GeneSHUTTLE enables us to prepare an improved liposome-DNA complex structure responsible for 100-fold enhanced gene delivery in vivo. The preparation of lipoplexes was performed under aseptic conditions according to the manufacturer's instructions. Briefly, in a microcentrifuge tube, 20 µl of 20 mM liposome were mixed with 30 µl of 5% dextrose in water (liquid 1). In a second microcentrifuge tube, 50 µg of ODN were mixed with 5% dextrose in water to produce a final volume of 50 µl (liquid 2). ODN (liquid 2) was mixed with liquid 1. This gives a final ODN concentration of 50 µg/100 µl with liposomes at 4 mM. Notably, it has been established that DNA, RNA, ribozymes, and oligonucleotides can all be delivered successfully with the liposomes (35). Finally, 200 µl of sterile distilled water containing synthetic double-stranded ODN (5 µg/g wt) were infused into the tail vein over 60 s at room temperature 60 min after LPS administration. This dose of NF-{kappa}B decoy ODN was chosen because our preliminary study showed that it was the minimum dose to cause constantly a >70% reduction in LPS-induced NF-{kappa}B activation in lung tissues. At the indicated time, mice were anesthetized with gaseous diethyl ether, the blood samples were collected by cardiac puncture for blood-gas analysis, and lungs were harvested, frozen immediately in liquid nitrogen, and stored at –80°C.

Gel mobility shift assay. Nuclear extracts were prepared from lung tissues of untreated and LPS-treated mice using methods described previously (24). In brief, mouse lung tissues were homogenized with a Polytron in 4 vol of ice-cold homogenization buffer [10 mM HEPES (pH 7.5), 0.5 M sucrose, 0.5 mM spermidine, 0.15 mM spermin, 5 mM EDTA, 0.25 M EGTA, 7 mM {beta}-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride]. After centrifugation at 12,000 g for 30 min at 4°C, the pellet was lysed and homogenized in 1 vol of ice-cold homogenization buffer containing 0.1% Nonidet P-40. It was then centrifuged at 12,000 g for 30 min at 4°C, and the pelleted nuclei were washed twice with ice-cold buffer containing 0.35 M sucrose. The nuclei were preextracted with 1 vol of ice-cold homogenization buffer containing 0.05 M NaCl and 10% glycerol for 60 min at 4°C, and the concentration of DNA was adjusted to 1 mg/ml. After the nuclear extract was pelleted at 12,000 g for 30 min at 4°C, the supernatant was brought to 45% (NH4)2SO4 and stirred for 30 min at 4°C. The precipitated protein was collected after 17,000 g for 30 min, resuspended in homogenization buffer containing 0.35 M sucrose, and stored in aliquots at –80°C.

NF-{kappa}B decoy ODN was labeled as a probe at the 3' end by means of a 3' end-labeling kit (Perkin Elmer Life Sciences, Boston, MA). After end-labeling was completed, 32P-labeled ODN was purified by application of a P-20 column. Binding reactions (10 µl) including the 32P-labeled probe (0.5–1 ng, 10,000–15,000 cpm) and 1 µg of polydeoxyinosinic acid (Sigma, St. Louis, MO) were incubated with nuclear extract for 30 min at room temperature and then loaded onto 5% polyacrylamide gel. The gels were subjected to electrophoresis, dried, and preincubated with parallel samples 10 min before the addition of the labeled probe. As a control, samples were incubated with excessive doses of cold NF-{kappa}B ODN, which resulted in the disappearance of signals. Gels were analyzed by autoradiography.

Total RNA extraction and Northern blot analysis. Total RNA was extracted from lung tissues by the guanidinium thiocyanate-phenol-chloroform method with Isogen (Nippon Gene, Toyama, Japan), which has been used routinely in our laboratory (18). RNA purity was determined by the ratio of optical density measured at 260 and 280 nm (OD260/OD280), and RNA quantity was estimated at OD260.

For Northern blot analysis, RNA, 20 µg/lane, was subjected to electrophoresis on agarose-formaldehyde gels and then transferred to a Hybond-N+ nylon membrane (Amersham, Little Chalfont, Buckinghamshire, UK). The membrane was prehybridized in prewarmed rate-enhanced hybridization buffer (Amersham) at 65°C for 60 min. Hybridization was done with a [32P]dCTP-labeled random-primed murine HDC, histamine H1 receptor, or iNOS or eNOS cDNA probe (19, 21, 22). We quantitated the expression of mRNA by counting the radioactivity using a bioimaging analyzer (Fujix BAS 2000; Fuji Photo Film, Tokyo, Japan), as described previously (18); this was standardized to the mRNA of the constitutively expressed protein {beta}-actin on the same filter to correct for any variability in gel loading.

Western blot analysis. Samples of tissue homogenate (5–20 µg) were run on SDS-PAGE (12.5% polyacrylamide gel) and electrotransferred to a polyvinylidine difluoride filter membrane. To reduce nonspecific binding, the membrane was preincubated for 60 min at room temperature in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4) containing 1% BSA. The membrane was then incubated for 60 min at 4°C with primary antibody recognizing HDC, H1 receptor, iNOS, eNOS, intracellular adhesion molecule-1 (ICAM-1), or actin. For HDC recognition, we used a mouse monoclonal antibody that was kindly provided by Dr. S. Tanaka (Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan). For recognizing H1 receptors, iNOS, eNOS, ICAM-1, and actin, we used the following commercially available antibodies: anti-human H1 receptor rabbit polyclonal antibody (Chemicon International, Temecula, CA), anti-rabbit iNOS mouse monoclonal antibody (Affinity BioReagents, Golden, CT), anti-human eNOS rabbit polyclonal antibody (Affinity BioReagents), anti-rat ICAM-1 goat polyclonal antibody (R&D Systems, Minneapolis, MN), and anti-chicken actin mouse monoclonal antibody (Ab-5; NeoMarkers, Fremont, CA). After an extensive washing with PBS containing 0.05% Tween 20, the membrane was incubated with a suitable secondary antibody coupled to horseradish peroxidase for 60 min at room temperature. The blots were washed twice in PBS-Tween 20 buffer and subsequently visualized with an enhanced chemiluminescence detection system (Amersham), exposed to X-ray film, and analyzed by NIH Image software produced by Wayne Rasband (National Institutes of Health, Bethesda, MD).

Evaluation of lung vascular leak. Lung vascular albumin permeation was assessed by intravenous injection of 10 µCi of 125I-labeled albumin 30 min before mice were killed. At the end of the experimental protocol, under anesthesia with gaseous diethyl ether, a polyethylene catheter (22G; JELCO; Critikon, Tampa, FL) was inserted into the trachea, bronchoalveolar lavage (BAL) was performed by repeatedly infusing and removing 2 ml of prewarmed saline, and the third drainage of effluent was kept as BAL fluid. Thirty min after 125I-albumin injection, blood samples were obtained from the atrium in the presence of heparin. The radioactivity of the samples was counted in a gamma counter. Permeability index (PI) was defined as follows: PI = BAL fluid (cpm/µl)/blood (cpm/µl).

In another series of experiments, mice were killed at 10 h after LPS administration, the chest was opened via a mid-line incision, the lungs were gently infused with sterile saline (~5 ml) from the heart for 30 s and carefully dissected from large airways, heart, and mediastinal structures, and excess fluid was absorbed with soft tissue paper. The lungs were immediately weighed in preweighed aluminum dishes (wet weight) and again after remaining overnight at 80°C in a drying oven (dry weight) for calculation of the wet-to-dry weight ratio.

Histopathology examination. The paraffin embedding procedures were performed by giving ~2 ml of 4% paraformaldehyde to the anesthetized animal through intratracheal infusion. The lungs were harvested, and then the specimens were sliced into 12-µm-thick sections. After deparaffinization, hematoxylin- and eosin-stained slides were prepared using standard methods.

Plasma histamine measurement. As previously reported (20), a blood sample was rapidly collected into chilled tubes containing EDTA-2Na and centrifuged (4°C) to separate the plasma. The plasma was stored at –80°C until the day of assay. The plasma histamine concentration was determined by use of a sensitive radioimmunoassay (Eiken Chemical, Tokyo, Japan).

Statistical analysis. Data are expressed as means ± SE. Statistical assessment of the data was made by Student's t-test or a repeated-measures one-way ANOVA followed by Bonferroni's multiple comparison test when appropriate. A P value of <0.05 was considered to be statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We initially ascertained whether in vivo transfection of NF-{kappa}B decoy ODN can effectively prevent the DNA binding of NF-{kappa}B in mouse lungs. As shown in Fig. 1, the binding activity of NF-{kappa}B, as assessed by gel mobility shift assay, was markedly increased in lung tissues from 10-h endotoxemic animals compared with controls. Transfection of NF-{kappa}B decoy ODN, but not of scrambled decoy ODN, via intravenous injection resulted in a decrease in the binding of NF-{kappa}B nearly to the control level in lung tissues from LPS-treated mice. These results encouraged us to study the potential utility of NF-{kappa}B decoy ODN strategy for the treatment of septic lung by in vivo transfection into lung tissues via intravenous injection.



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  		Fig. 1. Gel mobility shift assay for NF-{kappa}B activity in mouse lung tissues. The gel mobility shift assay used nuclear extracts from lung tissues of mice under different conditions. The induced NF-{kappa}B shift bands are indicated. Lane 1, free probe showed no detection of NF-{kappa}B translocation; lane 2, nuclear extracts from lungs of control mouse were incubated with a 32P-labeled NF-{kappa}B probe; lane 3, nuclear extracts were taken from lungs of the mouse at 10 h after lipopolysaccharide (LPS) treatment; lane 4, nuclear extracts were taken from lungs of the 10-h LPS-treated mouse that was transfected with NF-{kappa}B decoy oligodeoxynucleotide (ODN) 1 h after LPS treatment; lane 5, complete competition for increased binding of NF-{kappa}B in nuclear extract from septic lungs incubated with a 32P-labeled NF-{kappa}B probe was observed in the presence of a 100-fold excess of unlabeled NF-{kappa}B ODN; lane 6, detection of NF-{kappa}B activity in nuclear extract from septic lungs incubated with a 32P-labeled NF-{kappa}B probe remained unchanged by a 100-fold excess of scrambled sequence ODN. Data are representative of 5 separate experiments.

 
Table 1 summarizes the values for blood gases at 10 h after mice were given LPS or its vehicle (sterile saline). The arterial PO2 value was significantly reduced from the baseline at 10 h after LPS administration. Also, a marked fall in arterial PCO2 was observed at 10 h. There was a drastic decrease from baseline detected for base excess in LPS-treated mice. Arterial blood pH became essentially acidosis after injection of endotoxin. NF-{kappa}B decoy ODN, introduced in mice in vivo 60 min after LPS administration, resulted in an evident improvement of all of these reduced blood-gas parameters. This effect was not seen in the case of transfection of scrambled decoy ODN.


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  		Table 1. Effect of NF-{kappa}B decoy ODN or its scrambled form transfection on blood gases in mice that were rendered endotoxemic by an intravenous injection of LPS

 
The basal concentration of histamine in the plasma was 75 ± 57 nM (n = 5). Administration of LPS to the mouse profoundly elevated the concentration of histamine in the plasma (516 ± 200 nM at 10 h, n = 5). The LPS-induced increase in the plasma histamine concentration was strongly inhibited by in vivo transfection of NF-{kappa}B decoy ODN (147 ± 48 nM, n = 5) but not of scrambled decoy ODN.

Northern blot analysis showed that mouse lung tissues expressed a 2.7-kb transcript corresponding to HDC mRNA (Fig. 2). The transcript level of HDC was increased 10.2-fold at 10 h after LPS administration. Immunoblot analysis indicated the presence of two forms of HDC with different molecular sizes, 53 and 74 kDa, in mouse lungs (see Fig. 6). It is believed that the 74-kDa form of HDC is the cytosolic enzyme and the 53-kDa form is the particulate enzyme recognized as an active form (21). Densitometric analysis revealed an 8.3-fold increase in the active form of the HDC protein in lungs from 10-h endotoxemic animals compared with controls (n = 4). Transfection of NF-{kappa}B decoy ODN, but not of its mutated form, strongly inhibited the increases in gene and protein expressions of HDC in lung tissues from 10-h endotoxemic mice (Figs. 2 and 6).



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  		Fig. 2. Northern blot analysis of histidine decarboxylase (HDC) mRNA expression in lungs from mice that were vehicle treated, 10-h LPS treated, and 10-h LPS treated and transfected with NF-{kappa}B decoy ODN or its scrambled form. A: representative autograph of Northern blot analysis of HDC mRNA and {beta}-actin mRNA expression. HDC mRNA was detected as a single major band of 2.7 kb. {beta}-Actin was unaffected by LPS treatment. B: summary of quantification of densitometric measurement as ratio of HDC mRNA relative to {beta}-actin mRNA. Values are means ± SE; n = 5. ***Significant difference from control (P < 0.001). ###Significant difference from LPS treatment alone (P < 0.001).

 


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  		Fig. 6. Western blots showing inhibition by NF-{kappa}B decoy ODN transfection, but not by its scrambled form, of LPS-induced upregulation of HDC, histamine H1 receptor, iNOS, and ICAM-1 and downregulation of eNOS protein levels in mouse lungs. No apparent difference in {beta}-actin, which served as loading control, among groups was noted. The results are representative of at least 4 different experiments.

 
Expression of the transgene of the histamine H1 receptor, which migrated at 3.3 kb and 3.9 kb, was detected by Northern blot analysis (Fig. 3), as demonstrated in our previous reports (20, 22). After induction of sepsis by LPS injection, the transcript level of the H1 receptor was increased ~2.7- to 3.8-fold in lungs compared with that shown in controls. NF-{kappa}B decoy ODN treatment significantly lowered the increased H1-receptor mRNA level in the lung tissue from LPS-treated mice. On Western blots (see Fig. 6), the protein level of the H1 receptor was determined by the density of a single band migrating at 57 kDa as previously reported (22). The H1-receptor protein level was increased 3.2-fold by LPS challenge (n = 4), which was partially suppressed by NF-{kappa}B decoy ODN transfection (2.2-fold from control, n = 4). Transfection of mutated NF-{kappa}B decoy ODN had no effect on the LPS-induced increases in H1-receptor mRNA and protein (Figs. 3 and 6).



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  		Fig. 3. Northern blot analysis of histamine H1-receptor (R) mRNA expression in lungs from mice that were vehicle treated, 10-h LPS treated, and 10-h LPS treated and transfected with NF-{kappa}B decoy ODN or its scrambled form. A: representative autograph of Northern blot analysis of H1-receptor mRNA and {beta}-actin mRNA expression. H1-receptor mRNA was detected as 2 major bands of 3.3 kb and 3.9 kb. {beta}-Actin was unaffected by LPS treatment. B: summary of quantification of densitometric measurement as ratio of H1-receptor mRNA relative to {beta}-actin mRNA. Values are means ± SE; n = 5. ***Significant difference from control (P < 0.001). ###Significant difference from LPS treatment alone (P < 0.001).

 
When LPS was given to mice, the mRNA level of iNOS was elevated in lungs in a time-dependent manner, as detected by Northern blot analysis (Fig. 4). However, in vivo transfection of NF-{kappa}B decoy ODN, but not of its scrambled form, resulted in a marked decrease in iNOS mRNA to the level almost similar to that obtained in control lungs. On Western blots (see Fig. 6), the 130-kDa iNOS protein was detectable in control lungs, which was increased 12.6-fold by LPS. This LPS-induced increase in iNOS protein expression was suppressed by treatment with NF-{kappa}B decoy ODN (3-fold from control) but not with scrambled decoy ODN (12.1-fold from control, n = 4 for each).



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  		Fig. 4. Northern blot analysis of inducible nitric oxide synthase (iNOS) mRNA expression in lungs from mice that were vehicle treated, 6-h and 10-h LPS treated, and 10-h LPS treated and transfected with NF-{kappa}B decoy ODN or its scrambled form. A: representative autograph of Northern blot analysis of iNOS mRNA and {beta}-actin mRNA expression. iNOS mRNA was detected as a single major band of 4.4 kb. {beta}-Actin was unaffected by LPS treatment. B: summary of quantification of densitometric measurement as ratio of iNOS mRNA relative to {beta}-actin mRNA. Values are means ± SE; n = 5. ***Significant difference from control (P < 0.001). ###Significant difference from LPS treatment alone (P < 0.001).

 
In contrast to iNOS, mRNA and protein levels of eNOS showed a time-dependent decrease after LPS injection (Figs. 5 and 6). At 10 h after LPS administration, the relative amounts of mRNA and 140-kDa protein for eNOS were decreased to 53 ± 5% (n = 5) and 63 ± 4% (n = 4) of control, respectively. The decreases in eNOS mRNA and protein were significantly prevented by treatment with NF-{kappa}B decoy ODN but not with scrambled decoy ODN (Figs. 5 and 6).



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  		Fig. 5. Northern blot analysis of endothelial NOS (eNOS) mRNA expression in lungs from mice that were vehicle treated, 6-h and 10-h LPS treated, and 10-h LPS treated and transfected with NF-{kappa}B decoy ODN or its scrambled form. A: representative autograph of Northern blot analysis of eNOS mRNA and {beta}-actin mRNA expression. eNOS mRNA was detected as a single major band of 4.7 kb. {beta}-Actin was unaffected by LPS treatment. B: summary of quantification of densitometric measurement as ratio of eNOS mRNA relative to {beta}-actin mRNA. Values are means ± SE; n = 5. ***Significant difference from control (P < 0.001). ###Significant difference from LPS treatment alone (P < 0.001).

 
Intravenous LPS challenge caused an 18-fold increase in lung microvascular permeability, as assessed by the transpulmonary flux of radiolabeled albumin (Fig. 7). Treatment with the histamine H1-receptor antagonist diphenhydramine (5 mg/kg iv) or the NOS inhibitor NG-nitro-L-arginine (L-NNA; 5 mg/kg iv) 60 min after LPS challenge significantly but partially attenuated lung permeability compared with that shown in mice receiving LPS. Even when the two drugs were given together, the effect was not complete. However, transfection of NF-{kappa}B decoy ODN, but not of its scrambled form, 60 min after LPS administration resulted in much stronger protection against increased lung permeability than treatment with both diphenhydramine and L-NNA.



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  		Fig. 7. Effects of NF-{kappa}B decoy ODN or its scrambled form transfection, diphenhydramine (DPH) treatment, and NG-nitro-L-arginine (L-NNA) treatment on lung permeability in mice that were rendered endotoxemic by an intravenous injection of LPS. Pulmonary transcapillary albumin transit was used to assess alterations in lung permeability. Animals were transfected with NF-{kappa}B decoy ODN or its scrambled form or pretreated with DPH (5 mg/kg) and/or L-NNA (5 mg/kg) 60 min after LPS challenge and killed 10 h later. Values are means ± SE; n = 5. ***Significant difference from control (P < 0.001). ###Significant difference from LPS treatment alone (P < 0.001).

 
Furthermore, as a quantitative measure of fluid clearance in lungs, wet-to-dry weight ratios were evaluated in lungs removed from mice killed at 10 h after LPS administration (Fig. 8). Although a combination of diphenhydramine and L-NNA significantly but incompletely prevented the increase in the ratio induced by LPS, NF-{kappa}B decoy ODN, but not its scrambled form, reduced the ratio nearly to the level of nonendotoxemic controls.



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  		Fig. 8. Effect of NF-{kappa}B decoy ODN or its scrambled form transfection, DPH treatment, and L-NNA treatment on increased lung water in mice that were rendered endotoxemic by an intravenous injection of LPS. Animals were transfected with NF-{kappa}B decoy ODN or its scrambled form or pretreated with DPH (5 mg/kg) and/or L-NNA (5 mg/kg) 60 min after LPS challenge and killed 10 h later. Lungs were then harvested for determination of wet-to-dry weight ratios. Values are means ± SE; n = 5. ***Significant difference from control (P < 0.001). ###Significant difference from LPS treatment alone (P < 0.001).

 
Light microscopy findings showed that massive infiltration of inflammatory cells and thickening of the alveolar septum were observed in lungs from mice at 10 h after LPS administration compared with that shown in control lungs (Fig. 9). There were no significant light microscopic differences between lungs from septic and scrambled decoy ODN-transfected septic animals. In lungs from septic animals that had been transfected with NF-{kappa}B decoy ODN, architecture of alveoli was preserved and infiltration of inflammatory cells was strongly prevented.



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  		Fig. 9. Photomicrographs of lung tissues (hematoxylin-eosin stained; x100). A: normal pulmonary histology in control group. B: marked interstitial infiltration of inflammatory cells (blue arrow) and thickening of the alveolar septum (black arrow) in endotoxemic group 10 h after LPS administration. C: protection of the normal pulmonary histology in NF-{kappa}B decoy ODN-transfected endotoxemic group.

 
Finally, we assessed the effect of NF-{kappa}B decoy ODN transfection on LPS-induced lung inflammation. When leukocytes in BAL fluid were counted with a hemacytometer, cell counts increased dramatically in the animals at 10 h after LPS administration (73 ± 6 x 105 cells/ml, n = 3) compared with those seen in the control animals (7 ± 1 x 105 cells/ml, n = 3). This LPS-induced increase in BAL leukocyte counts was strongly inhibited by transfection of NF-{kappa}B decoy ODN (14 ± 2 x 105 cells/ml) but not of its scrambled form (74 ± 9 x 105 cells/ml, n = 3). Moreover, the 90-kDa ICAM-1 protein, which plays a central role in cell-cell contact-mediated immune responses (7) and the adherence of leukocytes to epithelial cells (17), was markedly increased by LPS challenge in the lung (Fig. 6). Densitometric quantification of the signal showed that the expression level of ICAM-1 was 8.7-fold higher in 10-h endotoxemic than in control lungs (n = 4). LPS-induced expression of ICAM-1 was unchanged with scrambled decoy ODN transfection (8.8-fold from control, n = 4) but was greatly suppressed by NF-{kappa}B decoy ODN transfection (2.1-fold from control, n = 4).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In the present study, our NF-{kappa}B decoy strategy showed a dramatic improvement of blood-gas derangements, pulmonary vascular hyperpermeability, and lung histopathology, including inflammation, in in vivo animal models of endotoxic shock. It has been shown that many of the genes (i.e., cytokines, ICAM-1, and iNOS) that have been implicated in endotoxic shock contain NF-{kappa}B binding sites in the promoter/enhancer region (3, 14). With particular clinical relevance, NF-{kappa}B binding activity has been found to be increased in patients with acute inflammation and sepsis and to be correlated with clinical severity and mortality (1, 5, 29). Under the experimental conditions established in our laboratory, NF-{kappa}B activity was markedly increased in lung tissues from mice after induction of sepsis with LPS as shown by the gel shift assay, and in vivo transfer of NF-{kappa}B decoy ODN greatly reduced the stimulation of NF-{kappa}B activity in septic lungs. This clearly indicates the validity of NF-{kappa}B decoy strategy by in vivo transfection of decoy ODN containing the NF-{kappa}B cis-element. Therefore, the results of the present study led us to propose that the inhibition of stimulated NF-{kappa}B activity in lungs by NF-{kappa}B decoy ODN results in the suppression of expression of key molecules that may play a pivotal role in the pathogenesis of septic lung.

As demonstrated in our previous study using endotoxemic rabbits (20, 21), plasma histamine levels were greatly increased in mice after induction of sepsis with LPS. This increase in the circulating level of histamine was associated with increased tissue expression of HDC, an enzyme that only forms histamine in mammals, during sepsis induction. In vitro transfection of NF-{kappa}B decoy ODN resulted in marked decreases in HDC mRNA and protein nearly to the nonseptic control level in lung tissues, leading to strong inhibition of elevated circulating histamine in septic animals. Furthermore, NF-{kappa}B decoy ODN treatment showed a significant inhibitory effect on superinduction of the histamine H1-receptor gene caused by endotoxin. These findings suggest that both HDC and H1-receptor genes are NF-{kappa}B-sensitive genes. Thus transcription of HDC and H1 receptor appears to be regulated by NF-{kappa}B in a direct or indirect way. However, because only the blockage of NF-{kappa}B activation was insufficient to suppress superinduction of the H1-receptor gene, the H1 receptor may have other transcriptional factors.

In our mouse model, endotoxin induced a tremendous increment of gene and protein expressions of iNOS in lung tissues. Because activation of the iNOS gene is dependent on binding of NF-{kappa}B to its consensus motifs in the iNOS promoter region, the iNOS gene is a target for NF-{kappa}B (41). As expected, inhibition of NF-{kappa}B nuclear translocation by NF-{kappa}B decoy ODN strongly prevented enhanced pulmonary expression of the iNOS gene. Similar to our findings obtained in endotoxin-induced septic mice, it has been demonstrated that protein expression of iNOS can be inhibited by treatment with NF-{kappa}B decoy ODN in the carrageenin-induced hind paw edema, an acute model of inflammation (12).

A contributory role for histamine and NO in increased pulmonary vascular permeation during septic shock has been documented (10, 34, 40). In this study, we found that treatment with diphenhydramine or L-NNA significantly but partially inhibited endotoxin-induced lung vascular permeability. This suggests that histamine and NO may be partly responsible for mediating increased lung vascular permeability following endotoxemia. To our surprise, the inhibition by NF-{kappa}B decoy ODN transfection of the increase in lung vascular permeability after endotoxemia was much more pronounced than that by a combination of diphenhydramine and L-NNA. We thus assume that, by activation of the NF-{kappa}B signaling pathway in lung tissues, endotoxin could induce transcription of several yet to be determined molecules that could be actually involved in pulmonary edema, in addition to HDC, H1 receptors, and iNOS.

In the present study, eNOS expression was found to be downregulated in lung tissues after endotoxemia, suggesting hindrance of physiological regulatory events with involvement of eNOS-based NO synthesis. In previous studies with cultured cells, downregulation of eNOS, triggered by increased NO production via LPS-induced iNOS expression, has been reported (11, 16, 32). Such negative feedback regulation of eNOS expression by enhanced iNOS-derived NO formation has been recently demonstrated in blood vessels taken from endotoxemic rabbits (21). The impaired expression of eNOS may be associated with endothelial histology injury (39). Because endothelial dysfunction could alter physiological regulation of blood flow distribution by interfering with metabolic vasodilation in states of limited O2 supply (38), such a endotoxin-induced endothelial disorder may well contribute to abnormalities in physiological function in septic lung failure. We observed that NF-{kappa}B decoy ODN treatment ameliorated eNOS downregulation induced by endotoxin, indicating that the blockade of NF-{kappa}B activation may offer therapeutic benefits in endothelial disorder in septic lung.

Here, we showed a novel therapeutic strategy to prevent the development of septic lung failure by transfecting decoy ODN to block the binding of the critical transcription factor NF-{kappa}B to its promoter sequence of the target genes, thereby inhibiting gene expression of key molecules necessary for increased lung vascular permeability. Because sepsis is a common cause of death and drug resistance is becoming a major medical problem, our findings may provide a useful therapeutic tool for treating septic shock. However, in vitro studies suggest that NF-{kappa}B plays a role as a survival factor, responsible in part for "turning on" genes that could block cell death by apoptosis (15). It is therefore of great importance to address a number of unavoidable issues, including safety and side effects, in ongoing studies.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported in part by a Grant-in-Aid for Scientific Research and for Exploratory Research from the Ministry of Education, Science, Sports, and Culture of Japan.


   ACKNOWLEDGMENTS
 
We thank Prof. Toshihiko Iwanaga and Dr. Hiroo Teramae for kind help in histology. We are also grateful to Mami Fujinaga and Somako Tone for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Hattori, Dept. of Pharmacology, Hokkaido Univ. Graduate School of Medicine, Sapporo 060–8638, Japan (E-mail: yhattori{at}med.hokudai.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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