INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: genistein; LPS; NF-B; JNK; lung injury

Inhibitors of protein tyrosine kinases (PTK) have been proposed to reduce lung injury (1) and lethal toxicity (2). However, the mechanisms responsible for the effects of PTK inhibitors remain obscure. A role for PTK in intracellular signals initiated by lipopolysaccharide (LPS) has been demonstrated. LPS stimulation rapidly increases tyrosine phosphorylation of a number of proteins, including the mitogen-activated protein kinase (MAPK) family (3). Nonreceptor PTK, such as p56^lyn, p58^hck, and p59^c-fgr, are involved in LPS-induced signaling processes (4).

NF-B is an essential transcription factor that regulates the gene expression of various cytokines, chemokines, growth factors, and cell-adhesion molecules (5). The most predominantly characterized NF-B complex is a p50-p65 heterodimer, which at rest is associated with an inhibitor protein, IB, and is retained in the cytoplasm. Most importantly, phosphorylation regulates the activity of the inhibitior protein, IB-, which dissociates from NF-B in the cytoplasm. The active NF-B can then translocate to the nucleus, where it binds to the NF-B motif of a gene promoter and functions as a transcriptional regulator. A p65-p50 heterodimer has been detected in LPS-stimulated macrophages and lung tissue (6). Recent evidence indicates that LPS-induced NF-B activation in lung tissue is associated with lung neutrophilia, epithelial permeability, and lipid peroxidation (6, 7). In vivo activation of NF-B, but not other transcription factors in alveolar macrophages from patients with acute respiratory distress syndrome (ARDS), has also been demonstrated (8). Therefore, activation of NF-B binding to various gene promoter regions appears to be a key molecular event in the initiation of LPS-induced pulmonary disease.

Cytokine-induced neutrophil chemoattractant (CINC) in the rodent models of lung injury, which is structurally and functionally similar to the human chemokine, interleukin-8 (IL-8), has been shown to play an important role in neutrophil migration into the lung (6). Its correlation with the severity of the process suggests a role in the induction of lung inflammation. In addition, there is an increase in the activity of matrix metalloproteinases (MMPs), including MMP-9. Extracellular matrix (ECM)-degrading enzymes are crucial for activated neutrophils to transverse subsequent ECM barriers after adhesion and for transendothelial cell migration, since these proteolytic enzymes digest most of the ECM components present in basement membranes and tissue stroma (9). A 92-kD gelatinase (MMP-9) was also detected in bronchoalveolar lavage (BAL) fluid in LPS-induced lung injury and in patients with ARDS (10). The major source of CINC and MMP-9 appears to be alveolar macrophages (7, 11). CINC and MMP-9 are produced in response to LPS, tumor necrosis factor- (TNF-), and interleukin- (IL-) (6, 12). These inflammatory mediators have been demonstrated to be regulated at the transcription level by NF-B (6, 13). PTK inhibitors have been shown to be effective in preventing LPS-induced NF-B activation as well as NF-B-dependent cytokine (IL-6, IL-8, and TNF-) production in vitro (14). However, effects of PTK inhibitors on NF-B activation and NF-B-dependent gene products, such as CINC and MMP-9, have not been investigated in the LPS-induced acute lung injury model. The objective of the present study was to examine whether genistein, a specific inhibitor of PTK, inhibits NF-B activation during acute lung injury induced by LPS and, if so, to enumerate the effects of inhibition of NF-B activation on LPS-induced proinflammatory gene products, such as CINC and MMP-9, and neutrophil influx into the lungs.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal Protocols

Four groups of specific pathogen-free male Sprague-Dawley rats (280-300 g) were used: (1) control rats received an intratracheal instillation of 0.5 ml of LPS-free saline (0.9% NaCl); (2) a saline-genistein group injected with genistein (50 mg/kg body weight, intraperitoneally) 2 h before intratracheal instillation of 0.5 ml of LPS-free saline (0.9% NaCl); (3) an LPS-treated group received an intratracheal instillation of 6 mg/kg body weight of LPS (Escherichia coli lipopolysaccharide, 055:B5; Sigma Chemical Co., St. Louis, MO) in 0.5 ml LPS-free saline; and (4) an LPS-genistein group injected with genistein (50 mg/kg body weight, intraperitoneally) 2 h before intratracheal instillation of 6 mg/kg body weight of LPS in 0.5 ml of LPS-free saline. For intratracheal instillation, rats were treated with enflurane anesthesia. The trachea then was exposed after a 1-cm midline cervical incision, and LPS or saline was injected intratracheally through a 24-gauge catheter. LPS or saline administration was immediately followed by three insufflations of 1 ml of air through the catheter and by rotating the animals to attempt to homogeneously distribute LPS or saline in the lungs. After a few minutes, the rats recovered from the anesthesia and were immediately placed in a chamber. Animals were killed 4 h after LPS treatment, and the following parameters were monitored: (1) total cell count, cell differential count, and measurement of protein content and LDH activity in BAL fluid; (2) DNA binding activity of NF-B in alveolar macrophages and lung tissue; (3) protein tyrosine phosphorylation in lung tissue; (4) CINC activity in BAL fluid and lung tissue; (5) MMP-9 activity in BAL fluid and the supernates of alveolar macrophage cultures. In addition, DNA binding activity was also determined at 2, 4, 14, or 24 h after LPS treatment to determine the kinetics of NF-B activation in lung tissue.

Isolation of BAL Cells, Lung Tissue, and Cell Counts

Four hours after LPS treatment, the rats were killed and BAL was then performed through a tracheal cannula with aliquots of 8 ml each using ice-cold Ca²⁺/Mg²⁺-free phosphate-buffered medium (145 mM NaCl, 5 mM KCl, 1.9 mM NaH₂PO₄, 9.35 mM Na₂HPO₄, and 5.5 mM dextrose; pH 7.4) for a total of 80 ml for each rat. The bronchoalveolar lavagate was centrifuged at 500 × g for 5 min at 4° C and cell pellets were washed and resuspended in phosphate-buffered medium. Cell counts and differentials were determined using an electronic Coulter counter with a cell sizing analyzer (Coulter Model ZBI with a channelizer 256; Coulter Electronics, Bedfordshire, UK) as described by Lane and Mehta (15). Red blood cells, lymphocytes, neutrophils, and alveolar macrophages were distinguished by their characteristic cell volumes (16). The recovered cells were 98% viable, as determined by trypan blue dye exclusion. After lavage, lung tissue was removed, immediately frozen in liquid nitrogen, and stored at 70° C.

Measurement of Total Protein and Lactate Dehydrogenase (LDH) Activity

To assess the permeability of the bronchoalveolar-capillary barrier, total protein was measured according to the method of Hartree (17), using bovine serum albumin as the standard. The LDH activity and total protein were measured in the first aliquot of the acellular BAL fluid. The activity of LDH, a cytosolic enzyme used as a marker for cytotoxicity, was measured at 490 nm using an LDH determination kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany). LDH activity was expressed as U/L, using an LDH standard.

Nuclear Extracts

Nuclear extracts were prepared by a modified method of Sun and coworkers (18). Lavage cells were resuspended in Dulbecco's modified Eagle's medium (DMEM; Mediatech, Washington, DC), supplemented with 5% fetal bovine serum (FBS) (HyClone, Logan, UT), 2 mM glutamine, and 1,000 units/ml penicillin-streptomycin. DMEM medium (5 ml), containing 5 × 10⁶ alveolar macrophages, was added to six-well plates and incubated at 37° C in a humidified atmosphere of 5% CO₂ for 2 h. The nonadherent cells were then removed with two 1-ml aliquots of DMEM. At the end of the incubation, adherent cells (> 95% alveolar macrophages) were harvested and then resuspended in hypotonic buffer A (100 mM HEPES, pH 7.9, 10 mM KCl, 0.1 M ethylenediaminetetraacetic acid [EDTA], 0.5 mM dithiothreitol [DTT], 1% Nonidet P-40, and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) for 10 min on ice, then vortexed for 10 s. Nuclei were pelleted by centrifugation at 12,000 rpm for 30 s. Nuclear extracts were also prepared from lung tissue by the modified method of Deryckere and Gannon (19). Aliquots of frozen tissue were mixed with liquid nitrogen and ground to powder using a mortar and pestle. The ground tissue was placed in a Dounce tissue homogenizer (Kontes Co., Vineland, NJ) in the presence of 4 ml of buffer A to lyse the cells. The supernate containing intact nuclei was incubated on ice for 5 min, and centrifuged for 10 min at 5,000 rpm. Nuclear pellets obtained from alveolar macrophages or lung tissue were resuspended in buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.42 M NaCl, 1 mM EDTA, and 0.5 mM PMSF) for 30 min on ice. The supernatants containing nuclear proteins were collected by centrifugation at 10,000 rpm for 2 min, and stored at 70° C.

Electrophoretic Mobility Shift Assay (EMSA)

Binding reaction mixtures (10 µl), containing 5 µg (4 µl) nuclear extract protein, 2 µg poly(dI-dC)·poly(dI-dC) (Sigma), and 40,000 cpm ³²P-labeled probe in binding buffer (4 mM HEPES, pH 7.9, 1 mM MgCl₂, 0.5 mM DTT, 2% glycerol, and 20 mM NaCl), were incubated for 30 min at room temperature. The protein-DNA complexes were separated on 5% nondenaturing polyacrylamide gels in 1× TBE buffer, and autoradiographed. Autoradiographic signals for activated NF-B were quantitated by densitometric scanning using an UltroScan XL laser densitometer (LKB, Model 2222-020; Bromma, Sweden) to determine the intensity of each band. The oligonucleotide used as a probe for EMSA was a double-stranded DNA fragment, containing the NF-B consensus sequence (5'-CCTGTGCTCCGGGAATTTC CCTGGCC-3'), labeled with [-³²P]dATP (Amersham, Buckinghamshire, UK), using DNA polymerase Klenow fragment (Life Technologies, Gaithersburg, MD). Cold competition was performed by adding 100 ng unlabeled double-stranded probe to the reaction mixture. Supershift assays were performed in alveolar macrophages and lung tissue with polyclonal antibodies to the NF-B proteins (p50 and p65) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Western Blot Analysis

Lung tissue homogenate samples (55 µg or 100 µg protein/lane for phosphorylated protein tyrosine, Jun N-terminal kinase [JNK] and CINC) or aliquots of acellular BAL fluid (70 µl/lane for CINC) were separated on a 10% or 20% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE), as described by Towbin and coworkers (20). Separated proteins were electrophoretically transferred onto nitrocellulose paper and blocked for 1 h at room temperature with Tris-buffered SAL containing 3% bovine serum albumin (BSA). The membranes were then incubated with an anti-mouse phosphotyrosine PY 20 antibody (1/1,000), anti-rabbit phospho-JNK/JNK antibody, or antiserum against rat CINC (1/200) at room temperature for 1 h. Antibody labeling of protein bands was detected with enhanced chemiluminescence (ECL) reagents according to the supplier's protocol.

Measurement of CINC in BAL Fluid

CINC concentrations in the first acellular BAL fluid were measured using a rat Gro/CINC-1 enzyme-linked immunosorbent assay (ELISA) system (Amersham).

Zymographic Analysis of MMP-9

The gelatinolytic activities in BAL fluid or the supernates of alveolar macrophage cultures were determined using zymography with gelatin copolymerized with acrylamide in the gel according to previously published methods (21). To obtain the supernates of alveolar macrophage cultures, lavage cells were resuspended in RPMI-1640 medium (Mediatech) containing 2 mM glutamine, 100 units/ml mycostatin without FBS. Aliquots of 1 ml, containing 10⁶ alveolar macrophages, were added to 24-well plates (Costar, Cambridge, MA) and incubated at 37° C in a humidified atmosphere of 5% CO₂ for 2 h. The nonadherent cells were then removed, and adherent cells were counted and further incubated in 1 ml RPMI medium. After a 24-h incubation, the supernatant was collected and filtered.

Aliquots of BAL fluid and the culture supernatants, normalized for an equal volume (8 µl) or amount of protein (8 µg), were electrophoresed on a 10% SDS-PAGE gel with 0.1% gelatin as a substrate without boiling under nonreducing conditions. After removing SDS with 2.5% Triton X-100 for 2 h, gels were incubated for 20 h at 37° C in 50 mM Tris-Cl (pH 7.4) containing 10 mM CaCl₂ and 0.02% NaN₃. The gels were then stained for 1 h in 7.5% acetic acid/10% propanol-2 containing 0.5% Coomassie Brilliant Blue G250 and destained in the same solution without dye. Positions of gelatinolytic activity are unstained on a darkly stained background. The clear bands on the zymograms were photographed on the negative (Polaroid's 665 film) and the signals were quantified by densitometric scanning using an UltroScan XL laser densitometer (LKB, Model 2222-020) to determine the intensity of MMP-9 activity as arbitrary densitometric units. To confirm MMP-9 activity, aliquots of BAL fluid were analyzed by Western blotting with anti-human MMP-9 monoclonal antibody, which was raised against MMP-9 secreted by human HT1080 fibrosarcoma cells (22) and cross-reacts with rat MMP-9 (23).

Statistical Analysis

Values were expressed as means ± standard errors. Data for total protein, LDH activity, CINC concentrations, and neutrophil differential count were compared among the groups by one-way ANOVA. Significance was accepted at a p value of < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Total Protein and LDH Activity in BAL Fluid

BAL protein contents (Figure 1A) and LDH activity (Figure 1B) in LPS-treated animals were significantly increased by 4.5- and 4.7-fold, respectively, compared with saline control animals, indicating that intratracheal treatment of rats with LPS induced acute lung injury (p < 0.05). However, genistein pretreatment significantly inhibited LPS-induced changes in lung injury parameters by 74% (p < 0.05). There were no significant differences in these parameters between saline-genistein animals and saline control animals (p < 0.05).

View larger version (11K): [in this window] [in a new window]   Figure 1.   Levels of total protein (A) and activity of LDH (B) in bronchoalveolar lavage fluid. The groups represent rats treated as follows: Saline, saline (intratracheally); Saline-Geni, saline (intratracheally) and a pretreatment with genistein (intraperitoneally); LPS, LPS (intratracheally); LPS-Geni, LPS (intratracheally) and a pretreatment with genistein (intraperitoneally). Values represent means ± SEM of results from five experiments. *Significant differences compared with saline, p < 0.05, and significant difference compared with the LPS group, p < 0.05.

NF-B Activation in Alveolar Macrophages and Lung Tissue

Figures 2A and 2B show NF-B activation in alveolar macrophages and lung tissue, respectively, identified 4 h after intratrachael instillation of saline or LPS, respectively. The DNA-binding activities of NF-B in alveolar macrophages from saline control animals and saline-genistein animals were essentially nondetectable. In LPS-treated animals, NF-B activation was enhanced by 10-fold compared with saline control animals. This enhancement was effectively depressed (85% inhibition) by a 2-h pretreatment with genistein. Greater activation of NF-B (17-fold) was shown in lung tissue from LPS-treated animals compared with saline control animals. Genistein resulted in substantial and consistent decreases (97%) in LPS-induced NF-B activation in lung tissue. In addition to genistein, AG 126 (a specific PTK inhibitor) also substantially inhibited LPS-induced NF-B activation in alveolar macrophages and lung tissue (data not shown). The kinetics of NF-B activation in lung tissue showed that the activity of DNA binding of NF-B peaked at 2 h after LPS treatment and progressively decreased at 4 to 24 h, but the inhibitory effect of genistein was maximal at 4 h after LPS treatment (Figure 2C). NF-B activation in lung tissue was not detectable at any time evaluated in the animals treated with saline or saline-genistein (data not shown).

View larger version (45K): [in this window] [in a new window]   View larger version (36K): [in this window] [in a new window]   View larger version (60K): [in this window] [in a new window]   Figure 2.   EMSA illustrating DNA-binding activity of NF-B to the NF-B motif in alveolar macrophages (A) and lung tissue (B). The groups represent rats treated as follows: Saline, saline (intratracheally); Saline-Geni, saline (intratracheally) and a pretreatment with genistein (intraperitoneally); LPS, LPS (intratracheally); LPS-Geni, LPS (intratracheally) and a pretreatment with genistein (intraperitoneally). Nuclear extracts were prepared from alveolar macrophages (5 × 106 alveolar macrophages) and lung tissue. Addition of 100 ng of unlabled cold competitor to the LPS samples successfully competed for NF-B binding and eliminated the specific band (A, lane 5, and B, lanes 9 and 10). EMSA showing the time course for NF-B activation in lung tissue from rats treated with LPS or LPS and genistein (C). Densitometry of NF-B bands on EMSA is expressed as arbitrary densitometric units. Values represent means ± SEM of results from five experiments. Supershift EMSA using nuclear proteins in alveolar macrophages (D) and lung tissue (E ) 4 h after LPS treatment. The addition of antibodies to p50 and more so to p65 caused supershifts, with a reciprocal decrease in the intensity of the NF-B band. The supershift bands are indicated by arrows.

The addition of the cold competitor eliminated the specific bands in the samples from LPS-treated animals, indicating that the band on the autoradiogram was specific for NF-B binding. To confirm the protein binding to the NF-B motif was due to NF-B (p50 and p65 heterodimers), supershift assays were performed. These data indicate that NF-B heterodimers (p50/p65) were activated in alveolar macrophages (Figure 2D) and lung tissue (Figure 2E) after LPS treatment, since supershift was detected by antibodies to p50 and more so to p65, with a reciprocal decrease in the intensity of the NF-B band.

Protein Tyrosine Phosphorylation in Lung Tissue

Western blotting with antiphosphotyrosine antibodies was employed in order to examine whether protein tyrosine phosphorylation in lung tissue was affected by LPS. Figure 3A shows that LPS treatment enhanced tyrosine phosphorylation of several proteins with molecular masses of approximately 46, 48, and 54 kD. Genistein had little effect in the saline-treated group. However, genistein pretreatment effectively inhibited LPS-induced protein tyrosine phosphorylation. It may be speculated that the phosphorylated 46- to 54-kD proteins belong to a group of mitogen-activated protein kinases, JNK. To determine JNK activation in the lung tissue from LPS-treated animals, Western blot analysis with a phospho-specific JNK antibody was employed. As shown in Figure 3B, LPS treatment resulted in phosphorylation and activation of JNK. Genistein substantially blocked LPS-induced JNK activation. JNK activation was not detectable in the animals treated with saline or saline-genistein.

View larger version (18K): [in this window] [in a new window]   Figure 3.   Protein tyrosine phosphorylation (A) and activation of JNK (B) in lung tissue. The groups represent rats treated as follows: Saline, saline (intratracheally); Saline-Geni, saline (intratracheally) and a pretreatment with genistein (intraperitoneally); LPS, LPS (intratracheally); LPS-Geni, LPS (intratracheally) and a pretreatment with genistein (intraperitoneally). Western blots with antiphosphotyrosine antibodies or anti-phospho-JNK/JNK antibody were employed to monitor protein tyrosine phosphorylation or JNK phosphorylation. In A, molecular sizes are indicated in kilodaltons and arrows indicate protein bands whose phosphorylation is altered. Phosphorylation signals and protein abundance signals of JNK are presented in B. Results are representative of five independent experiments.

CINC and MMP-9 Production in Alveolar Macrophages or Lungs

CINC and MMP-9 were chosen in our experiments as representative inflammatory mediators because of their important roles in neutrophil influx and lung damage and because their gene regulation is dependent on NF-B. Figures 4A and 4B illustrate representative Western blots of lung tissue and BAL fluid for CINC, respectively. CINC protein expression was detectable in the samples of saline control animals, but was markedly increased by LPS treatment for 4 h. Genistein decreased the level of LPS-induced CINC expression in lung tissue and BAL fluid. ELISA results showed that CINC protein levels in BAL fluid increased by 27-fold in BAL fluid (Figure 4C). Genistein inhibited this LPS-induced elevation of CINC levels by 43%. Genistein alone had little effect on CINC levels in lavage fluid.

View larger version (18K): [in this window] [in a new window]   Figure 4.   CINC activity in lung tissue and bronchoalveolar lavage fluid. The groups represent rats treated as follows: Saline, saline (intratracheally); Saline-Geni, saline (intratracheally) and a pretreatment with genistein (intraperitoneally); LPS, LPS (intratracheally); LPS-Geni, LPS (intratracheally) and a pretreatment with genistein (intraperitoneally). Western blot with anti-CINC antibodies was performed on the samples of lung tissue (A) and BAL fluid (B). Results are representative of five independent experiments. CINC concentrations in BAL fluid (C). CINC protein levels were quantified by enzyme-linked immunosorbent assay. Values represent means ± SEM of results from five experiments. *Significant differences compared with saline, p < 0.05, and significant difference compared with the LPS group, p < 0.05.

As shown in Figures 5A and 5B, BAL fluid and the supernates from alveolar macrophage cultures were analyzed for evidence of MMP-9 activity using gelatin zymography. The BAL fluid from saline control animals showed only a weak 66-kD gelatinolytic band, which is a molecular weight identical to MMP-2 and no detectable 92-kD gelatinolytic band, which is a molecular weight identical to MMP-9 (21). LPS treatment induced a distinct increase in the amount of gelatinolytic activity and the most prominent band was a 92-kD species (MMP-9) in BAL fluid. Its identity was confirmed as MMP-9 by Western blot analysis with the anti-MMP-9 monoclonal antibody (Figure 5C, lane 2). By densitometric analysis, MMP-9 activity in BAL fluid from LPS animals was approximately 9.5-fold higher than that from saline control animals. In the supernates from alveolar macrophage cultures of saline control animals, MMP-9 activity was also not detectable, but was significantly increased by 3.4-fold in the sample from LPS animals. A pretreatment with genistein effectively inhibited LPS-induced MMP-9 activity by 70% and 63% in BAL fluid and the supernates from alveolar macrophage cultures, respectively. MMP-9 activity was not detectable at all in the samples from saline-genistein animals.

View larger version (25K): [in this window] [in a new window]   Figure 5.   Gelatinolytic activities in bronchoalveolar lavage fluid (A) and the supernates from alveolar macrophages in culture (B). The groups represent rats treated as follows: Saline, saline (intratracheally); Saline-Geni, saline (intratracheally) and a pretreatment with genistein (intraperitoneally); LPS, LPS (intratracheally); LPS-Geni, LPS (intratracheally) and a pretreatment with genistein (intraperitoneally). Alveolar macrophages (106/ml of RPMI medium) were incubated for 24 h. BAL fluid and culture supernates were analyzed by a sensitive zymography followed by scanning densitometry. The 92-kD and 66-kD gelatinolytic bands correspond to MMP-9 and MMP-2, respectively. Densitometry of 92-kD bands is expressed as arbitrary densitometric units. Values represent means ± SEM of results from five and four experiments for bronchoalveolar lavage fluid and the supernates from alveolar macrophages in culture, respectively. Western blot of bronchoalveolar lavage fluid from saline and LPS group with an antihuman-MMP-9 monoclonal antibody (C).

Neutrophil Influx into Lungs

BAL cells were differentially analyzed to evaluate the effects of inhibition of NF-B activation by genistein on LPS-induced neutrophil influx. As shown in Figure 6, neutrophils accounted for 41% of the total lung lavage cells (29.5 × 10⁶) in LPS-treated animals, indicating a significant increase in neutrophil influx into the alveolar spaces compared with saline control animals (3% of the total lung lavage cells [11.1 × 10⁶], p < 0.05). Genistein significantly suppressed the percentage of BAL neutrophils to 12% of the total lung lavage cells ([12.5 × 10⁶], versus LPS animals, p < 0.05). The total lung lavage cells and percentage of BAL neutrophils in saline-genistein animals were not significantly different compared with saline control animals.

View larger version (10K): [in this window] [in a new window]   Figure 6.   Neutrophil differential counts (%) in bronchoalveolar lavage fluid. The groups represent rats treated as follows: Saline, saline (intratracheally); Saline-Geni, saline (intratracheally) and a pretreatment with genistein (intraperitoneally); LPS, LPS (intratracheally); LPS-Geni, LPS (intratracheally) and a pretreatment with genistein (intraperitoneally). Values represent means ± SEM of results from five experiments. *Significant difference compared with saline, p < 0.05, and significant difference compared with the LPS group, p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study, we determined (1) the in vivo relation between NF-B activation and LPS-induced acute lung injury; (2) the inhibition of protein tyrosine phosphorylation, JNK activation, NF-B activation, and acute lung injury by genistein; and (3) the effects of inhibiting NF-B activation on LPS-induced proinflammatory gene products, such as CINC and MMP-9, as well as neutrophil influx into the lungs. The activation of NF-B has been associated with lung injury in LPS- and silica-treated rats (6, 7, 24) and patients with ARDS (8). Data from our laboratory indicate that in vivo activation of NF-B in LPS-treated rats preceded the transcription of genes for proinflammatory mediators (7) or lung inflammation and injury, that is, the increases in neutrophil numbers, total protein, and LDH activity in BAL fluid (data not shown). These results suggest that NF-B is an important intracellular target for the early detection and prevention of lung injury. Consistent with our kinetic results, Blackwell and coworkers (6) reported that NF-B activation in lung tissue peaked by 2 h after LPS treatment (6 mg/kg, intraperitoneally). However, in their study, NF-B activity dissipated by 4 to 6 h after LPS treatment. In contrast, in our study, it was still 17-fold greater than control at 4 h after LPS treatment (6 mg/kg, intratracheally) and remained detectable at 14-24 h after LPS treatment. The disparity may in part be related to differences in the route of administration of LPS. In addition to NF-B activation in lung tissue, we also found increases in DNA-binding activity of NF-B in alveolar macrophages.

The mechanisms by which genistein downregulates LPS- induced NF-B activation have not been resolved. LPS stimulation has been shown to rapidly increase protein-tyrosine phosphorylation of a number of proteins in vitro (3). Consistent with in vitro data, we reported here that in vivo treatment with LPS for 4 h enhanced tyrosine phosphorylation of several proteins with molecular masses of approximately 46, 48, and 54 kD. This event was prevented by pretreatment with genistein. These data indicate that LPS could trigger PTK activation in lung cells, and suggest the involvement of PTK in LPS-induced NF-B activation in vivo. A role of PTK in the activation of NF-B has been suggested for monocytes, macrophages, and T cells after in vitro exposure to LPS, cytokines, or silica (14, 25). However, there has been no evidence to date that these signal transductions are linked to the pathological conditions. Our in vivo experiments clearly indicate that genistein attenuates acute lung injury induced by LPS in addition to inhibiting tyrosine phosphorylation and NF-B activation. This is the first demonstration to suggest a role of PTK in NF-B activation in an animal model of LPS-induced acute lung injury. What type of PTK is involved in the NF-B activation is not clear. Several lines of evidence suggest that members of the src family of PTK, such as hck, fgr, and lyn, play important roles in macrophage activation by LPS. In addition, LPS rapidly induces tyrosine phosphorylation of the MAPK family. In agreement with the molecular masses expressed as tyrosine phosphorylated proteins, phosphorylated JNK was detected in lung tissue from LPS-treated animals in the present study. Genistein substantially prevented this JNK activation. We have also found that genistein and/or src family inhibitors inhibit LPS-induced NF-B activation and ERK or JNK phosphorylation in RAW 264.7 macrophages (data not shown). Involvement and interconnection of MEK-1 (MAP kinase/Erk kinase) and IB kinase  in NF-B activation have been demonstrated (5). Furthermore, Briant and coworkers (29) have reported that MEK-1 and ERK act as intermediates in the cascade of events that regulate NF-B activation. Therefore, it is possible that a genistein-sensitive src family of PTK is responsible for activation of members of the MAPK family linked to NF-B activation associated with acute lung injury.

The data from the present study indicate that the inhibition of LPS-induced NF-B activation by genistein may explain the inhibition of downstream LPS-induced NF-B-dependent responses. These include proinflammatory gene products, adherence, migration, and activation of neutrophils, and cytotoxity (2, 14, 30). Our findings indicate an increase in LPS-induced CINC production in lung tissue and BAL fluid. Its correlation with NF-B activation and lung injury is consistent with recent in vivo studies. The contribution of CINC to the development of lung inflammation and injury was demonstrated in LPS-, IgG immune complex-, or ozone-exposed rats (6, 31, 32). Blackwell and coworkers (6, 33) and Liu and coworkers (7) have reported that LPS-induced NF-B activation was temporally correlated with the expression of CINC mRNA in lung tissue.

Recently, MMPs have been implicated in the progression of acute lung injury and repair, due to their ability to cleave all the proteins constituting ECM (9). In the present study, highly increased MMP activities were observed in BAL fluid of LPS-treated animals with the most prominent enzyme being MMP-9. In alveolar macrophages, only MMP-9 was markedly increased. The increases in MMP-9 were correlated with neutrophil influx and lung injury. Accordingly, increases in gelatinase activities including MMP-9 and MMP-2 have been reported in BAL fluid and/or the supernates of alveolar macrophages of IgA- or LPS-exposed animals (11) and patients with ARDS (10) and asthma (34).

The present study is the first to demonstrate that genistein caused effective reduction of LPS-induced CINC production and MMP-9 activity consistent with suppression of neutrophil influx into the lung. These results support the concept of a correlation between inhibition of NF-B and suppression of the inflammatory response.

In conclusion, data from the present study suggest that genistein inhibits an upstream tyrosine kinase pathway. This inhibition would effectively block amplification of the LPS-induced catalytic cascade. Furthermore, tyrosine phosphorylation itself and various NF-B-dependent proinflammatory cytokines strongly induce the DNA-binding activity of NF-B. Therefore, genistein may block the NF-B-mediated positive feedback of uncontrolled inflammation and lung injury. Genistein has a very low toxicity in animal studies (35). We propose that genistein may have potential as a treatment in the early stages of lung injury, to attenuate the inflammatory cascade closely associated with NF-B activation. Further research is warranted to evaluate the therapeutic potential of genistein.