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Published ahead of print on April 30, 2003, doi:10.1164/rccm.200209-1051OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 85-91, (2003)
© 2003 American Thoracic Society

Original Article

ß-Lapachone Reduces Endotoxin-induced Macrophage Activation and Lung Edema and Mortality

Huei-Ping Tzeng, Feng-Ming Ho, Kuo-Fang Chao, Min-Liang Kuo, Shoei-Yn Lin-Shiau and Shing-Hwa Liu

Institute of Toxicology, College of Medicine, National Taiwan University, Taipei; and Department of Nursing, Chung-Tai Institute of Health Sciences and Technology, Taichung, Taiwan

Correspondence and requests for reprints should be addressed to Shing-Hwa Liu, Ph.D., Institute of Toxicology, College of Medicine, National Taiwan University, Number 1, Jen-Ai Road, Section 1, Taipei 10043, Taiwan. E-mail: shliu{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ß-Lapachone, a 1,2-naphthoquinone, is a novel chemotherapeutic agent. It has been shown to be capable of suppressing inducible nitric oxide synthase expression and function in rat alveolar macrophages. The authors further performed experiments to examine the molecular mechanism of ß-lapachone on LPS-induced responses in rat alveolar macrophages and to evaluate its in vivo antiinflammatory effect. A significant increase in nitrite production and inducible nitric oxide synthase expression was elicited in macrophages treated with LPS that was inhibited by coincubation with ß-lapachone. ß-Lapachone could also inhibit the production of tumor necrosis factor-{alpha} induced by LPS. LPS induces protein tyrosine phosphorylation and nuclear factor-{kappa}B binding activity by gel mobility shift assay in macrophages. These events were significantly inhibited by ß-lapachone. Furthermore, ß-lapachone in vivo protected against the induction of lung edema, lung-inducible nitric oxide synthase protein expression and nuclear factor-{kappa}B activation, lethality, and increased plasma nitrite and serum tumor necrosis factor-{alpha} levels induced by LPS. These results indicate that ß-lapachone suppresses inducible nitric oxide synthase induction and tumor necrosis factor-{alpha} production mediated by the inhibition of protein tyrosine phosphorylation and nuclear factor-{kappa}B activation caused by LPS. This results in a beneficial effect in an animal model of sepsis.

Key Words: ß-lapachone • inducible nitric oxide synthase • tumor necrosis factor-{alpha} • nuclear factor-{kappa}B • protein tyrosine phosphorylation

ß-Lapachone, a 1,2-naphthoquinone, and its derivatives have been shown to exhibit a number of pharmacologic actions, including antiviral, antiparasitic, and antitumor activities (1–4). Some studies have shown that ß-lapachone was capable of inducing human carcinoma cells, including lung cancer cells, to undergo programmed cell death (5, 6), but not in normal human lymphocytes (6). ß-Lapachone has been identified to be a potent candidate as a topoisomerase I (3) and II (7) inhibitor. It has recently been considered that ß-lapachone was one of a few novel anticancer drugs currently under active investigation, and it showed promise for chemotherapy alone and especially in combinations (2). The topoisomerase I inhibitor topotecan has been identified for recent development as a new oral chemotherapeutic agent for lung cancer (37). Recently, we demonstrated that ß-lapachone, in response to gram-negative bacterial LPS, prevents the induction of inducible nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages and isolated vessels (8). However, the molecular mechanism of action of ß-lapachone on LPS-induced responses in macrophages and its in vivo evaluation need further investigation.

LPS is released during bacterial infection and is a potent activator of monocytes and macrophages. LPS triggers the production of a variety of proinflammatory cytokines, such as tumor necrosis factor-{alpha} (TNF-{alpha}), as well as excessive amounts of NO, which have a key role in inflammatory and immune responses (9, 10). In the alveolar fluid, LPS can bind to soluble LPS-binding protein and is then transferred to CD14 expressed on alveolar macrophages and bronchial epithelial cells (11) and, in conjunction with MD-2 signals via, at least, Toll-like receptor 4. After binding to these receptors, LPS induces many intracellular events, such as the activation of protein tyrosine kinases and mitogen-activated protein kinase signaling pathways (12). The cascade reaction of these kinases will lead to the activation and the induction of nuclear factors. The LPS signaling pathway leading to TNF-{alpha} production is unknown but has been reported to depend on the activation of protein tyrosine kinases, the proto-oncogene product Ras, and the serine-threonine kinase Raf-1 (13). The TNF-{alpha} gene is regulated by various transcription factors, including nuclear factor-{kappa}B (NF-{kappa}B)/Rel proteins and Egr-1 (14, 15). The promotor region of the murine gene for iNOS has also been characterized (16), and the activation of NF-{kappa}B has been shown to mediate the enhanced expression of the iNOS gene in macrophages exposed to LPS (17). Thus, we performed experiments to test the hypothesis of possible involvement of protein tyrosine phosphorylation and NF-{kappa}B signaling in the inhibition of ß-lapachone on LPS-induced iNOS and TNF-{alpha} production in macrophages. The in vivo effects of ß-lapachone on LPS-induced lung edema, mortality, and lung extracellular-regulated kinase (ERK1/2) phosphorylation and NF-{kappa}B signaling were also evaluated.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures
The primary cell culture of rat alveolar macrophages and mouse monocyte/macrophage cell line RAW264.7 (ATCC-TIB71) were used for studies. Alveolar macrophages were obtained by modification of the bronchoalveolar lavage method as previously described (18). Wistar rats (200–250 g) were anesthetized using sodium pentobarbital before lavage. The cells were resuspended in RPMI-1640 medium with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified incubator with 5% CO2.

Nitrite/nitrate Assay
Nitrite/nitrate in the cell culture supernatant or mouse plasma was determined by using the colorimetric assay kit contained nitrate reductase (R&D Systems, Minneapolis, MN).

Cytotoxicity Assay
Cytotoxicity was performed by the tetrozolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl-tetrazolium bromide colorimetric assay. The data for cytotoxicity were obtained by reading the plates on a Dynatech-MR5000 multiwell scanning spectrophotometer at a wavelength of 570 nm.

Western Blot Analysis
Total protein containing 30–50 µg of cellular lysates protein or nuclear extracts of alveolar macrophages was separated on 8% sodium dodecyl sulfate-polyacrylamide minigels and transferred to nitrocellulose membranes. After blocking, blots were incubated with anti-inhibitor {kappa}B{alpha} (I{kappa}B{alpha}), NF-{kappa}B-p65, phospho-ERK1/2, {alpha}-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), or phosphotyrosine (BD Transduction Laboratories, San Diego, CA) antibodies in phosphate-buffered saline (PBS)/Tween-20 for 1 hour followed by two washes in PBS/Tween-20 and then incubated with horseradish peroxidase-conjugated goat antimouse IgG for 30 minutes. The antibody-reactive bands were revealed by the enhanced chemiluminescence kit (Amersham, Bucks, UK).

Immunoprecipitation
The cellular lysates were incubated with 4 µL of anti-ERK1/2 antibody on ice for 4 hours. Immune complexes were precipitated with protein A-agarose conjugate and washed five times with 1 ml of ice-cold lysis buffer. Bound proteins were eluted by heating the samples with 60 µL of 2 x sodium dodecyl sulfate sample buffer for 5 minutes at 95°C.

Electrophoretic Mobility Shift Assays
Nuclear extracts of alveolar macrophages were prepared and assayed as previously described (19); 5 µg of each nuclear extract was mixed with the labeled double stranded NF-{kappa}B oligonucleotide and incubated at room temperature for 20 minutes. Radioactive bands were detected by autoradiography.

Assay for TNF-{alpha}
TNF-{alpha} in cell culture supernatant or mouse serum was assayed using a mouse TNF-{alpha} ELISA kit (R&D Systems). The minimum detectable dose is typically less than 5.1 pg/ml.

Measurement of Lung Edema
BALB/c mice (20–25 g), provided by the Laboratory Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan), would be used as our in vivo experimental animals. The Animal Care Committee of the College of Medicine, National Taiwan University, conducted the study in accordance with the guideline for the care and use of laboratory animals. Mice were sacrificed by ether anesthesia, and lungs were excised. All extrapulmonary tissue was cleared, weighed (wet weight), dried for 48 hours at 60°C, and weighed again (dry weight). Lung edema was expressed as the ratio of wet weight to dry weight.

Materials
ß-Lapachone was prepared according to the procedures as previously described (3). ß-Lapachone was dissolved in dimethylsulfoxide (DMSO). LPS (Escherichia coli-026:B6) was obtained from Sigma Chemical (St. Louis, MO).

Statistics
Data are expressed as means ± SEM. Statistical analysis was performed using one-way analysis of variance followed by Dunnett's test for each paired experiment (20); p values of less than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of ß-Lapachone on iNOS Protein Expression and TNF-{alpha} Release
Rat alveolar macrophages and RAW264.7 cells were used to study the effects of LPS or combination with ß-lapachone on nitrite production and iNOS protein expression. The exposure of macrophages to LPS (10 µg/ml) for 24 hours was associated with the increase of nitrite production and iNOS protein expression, which was inhibited by coincubation with ß-lapachone (1–4.5 µM) (Figures 1A and 1B) . Under the same conditions, there was no cytotoxicity in alveolar macrophages and RAW264.7 cells using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl-tetrazolium bromide assay (Figure 1C). Moreover, the TNF-{alpha} secreted from alveolar macrophages into the cell medium was determined. LPS induced a marked increase in TNF-{alpha} levels, which was significantly inhibited by ß-lapachone (1–4.5 µM) (Figure 2) .



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  		Figure 1. Inhibition of nitrite production and the iNOS protein expression in macrophages stimulated with LPS by ß-lapachone. Rat alveolar macrophages (AM) and RAW264.7 cells were stimulated with LPS (10 µg/ml) in the presence or absence of ß-lapachone (ß-LP, 1–4.5 µM) for 24 hours, and then the nitrite production (A) and iNOS protein expression (B) were measured. The cytotoxicity of ß-lapachone in macrophages in the presence or absence of LPS was also determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide assay, as described in the METHODS (C). Data are presented as means ± SEM of three to five separate experiments performed in triplicate. *p < 0.05 as compared with LPS alone. (B) Results are representative of at least three independent experiments.

 


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  		Figure 2. Effect of ß-lapachone on LPS-induced production of TNF-{alpha} from alveolar macrophages. Rat alveolar macrophages were stimulated with LPS (10 µg/ml) in the presence or absence of ß-lapachone (ß-LP, 1–4.5 µM) for 24 hours. Data are presented as means ± SEM of three to five separate experiments performed in triplicate. *p < 0.05 as compared with LPS alone.

 
Inhibition by ß-Lapachone of LPS-induced Degradation of I{kappa}B{alpha} and NF-{kappa}B Activation
Activation of NF-{kappa}B involves the degradation of the inhibitory subunit, I{kappa}B{alpha}, from a cytoplasmic complex that consists of NF-{kappa}B and I{kappa}B{alpha} (21). To examine the involvement of ß-lapachone in the prevention of the degradation of I{kappa}B{alpha} protein, immunoblot analysis of I{kappa}B{alpha} protein was performed. As shown in Figure 3A, I {kappa}B{alpha} disappeared from the NF-{kappa}B complex in alveolar macrophages stimulated with LPS for 30 minutes, which was prevented in the presence of ß-lapachone (3 µM). To investigate further whether the ß-lapachone was capable of inhibiting the activation of NF-{kappa}B, the levels of NF-{kappa}B p65 protein in the nuclei of macrophages were investigated with immunoblotting. Furthermore analysis of NF-{kappa}B binding activity by gel mobility shift assay was performed. The result showed a marked NF-{kappa}B p65 protein expression in the nuclei at 1 hour after LPS treatment. ß-Lapachone (3 µM) effectively abolished this LPS-triggered NF-{kappa}B nuclear translocation (Figure 3B). In subsequent experiments, we determined NF-{kappa}B binding activity at 1 hour after LPS treatment. As shown in Figure 3C, the induction of specific NF-{kappa}B binding activity by LPS was effectively inhibited by ß-lapachone (3 µM). The addition of 50-fold excess unlabeled NF-{kappa}B oligonucleotide, which would compete for the binding of NF{kappa}B, completely prevented the band shifts, demonstrating the specificity of the protein/DNA interaction (Figure 3C, lane 5). Supershift analysis has shown that the induced NF-{kappa}B was a p65/p50 heterodimer (data not shown).



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  		Figure 3. Inhibition of LPS-induced I{kappa}B{alpha} degradation and NF-{kappa}B binding activity by ß-lapachone. Rat alveolar macrophages were stimulated with LPS (10 µg/ml) in the presence or absence of ß-lapachone (ß-LP, 3 µM) for 30–60 minutes. After 30 minutes of incubation, cytosolic fractions were prepared and analyzed for the content of I{kappa}B{alpha} protein by Western blot. {alpha}-Tubulin served as the control for sample loading and integrity (A). After 1 hour of incubation, the nuclear extracts were prepared for analysis of NF-{kappa}B p65 protein by Western blot (B) and NF-{kappa}B binding activity by electrophoretic mobility shift assay (C) as described in the METHODS. The specificity of the binding was determined by adding 50-fold molar excess unlabeled NF-{kappa}B (lane 5). Results shown are representative of at least three independent experiments.

 
Inhibition of ß-Lapachone on LPS-stimulated Protein Tyrosine Phosphorylation
Tyrosine phosphorylation pathways are essential components of the process of macrophage activation and the resultant production of inflammatory mediators such as NO and TNF-{alpha} (13, 22). We therefore examined the effect of ß-lapachone on protein tyrosine phosphorylation using the method of antiphosphotyrosine immunoblot. Tyrosine phosphorylation of multiple proteins induced by LPS was detected 5 minutes after stimulation and continued to increase over a period of 30 minutes. The results are illustrated in Figure 4A . ß-Lapachone (3 µM) was capable of inhibiting the LPS-stimulated protein tyrosine phosphorylation. Furthermore, we performed immunoprecipitation using antibody against ERK1/2; subsequently, the immune complex was subjected to Western blot analysis using an antiphosphotyrosine antibody. As shown in Figure 4B, LPS stimulated ERK1/2 tyrosine phosphorylation, which was effectively inhibited by ß-lapachone.



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  		Figure 4. Inhibition of LPS-induced protein tyrosine phosphorylation by ß-lapachone. Rat alveolar macrophages were stimulated with LPS (10 µg/ml) in the presence or absence of ß-lapachone (ß-LP, 3 µM) for various time intervals (5–60 minutes). Tyrosine phosphorylation (P-Tyr) of multiple proteins induced by LPS was detected 5 minutes after stimulation and continued to increase over 30 minutes (A). After a 15-minute incubation, whole-cell lysates immunoprecipitated with anti-ERK1/2 antibody were blotted with antiphosphotyrosine antibody (B). Results shown are representative of at least three independent experiments. IP = immunoprecipitation; WB = western blot.

 
In Vivo Effects of ß-Lapachone on LPS-induced Responses
We evaluated the effect of ß-lapachone in attenuating the LPS-induced lung edema and mortality in vivo. Mice were given vehicle (DMSO) or ß-lapachone (1.25, 2.5, or 5 mg/kg, intraperitoneally) simultaneously with, or 1 h after, the treatment of LPS (7.5 mg/kg for lung edema test; 25 mg/kg for lethal toxicity test, intraperitoneally). A significant lung edema was produced, as detected by an increase in the wet/dry weight ratio when mice were treated with LPS (7.5 mg/kg) for 6 hours. ß-Lapachone (2.5 and 5 mg/kg) effectively antagonized the LPS-induced lung edema in mice (Figure 5A) . To investigate further the induction of iNOS in lungs of LPS-treated mice with or without the treatment of ß-lapachone, Western blot analysis was used. The results showed that LPS elicited an induction in iNOS protein in lungs, which could be inhibited by ß-lapachone (2.5 mg/kg) (Figure 5B). Moreover, mice were intraperitoneally given LPS at a dose of 25 mg/kg, induced 80% lethality within 96 hours (Figure 6) . ß-Lapachone (2.5 mg/kg) was effective in reducing the LPS-induced mortality (Figure 6). Mice receiving DMSO (vehicle) or ß-lapachone alone exhibited no lung edema and lethality. On the other hand, intraperitoneal injection of mice with LPS (7.5 mg/kg) significantly increased the total plasma nitrite levels as compared with control mice at 6 hours (Figure 7A) . This increase was significantly inhibited by ß-lapachone (2.5 mg/kg, intraperitoneally), which was administrated simultaneously with, or 1 hour after, the application of LPS (Figure 7A). Moreover, mice, which were intraperitoneally given LPS (7.5 mg/kg), induced a marked increase in serum TNF-{alpha} levels, reaching a peak after approximately 1 hour (Figure 7B). Simultaneous treatment with ß-lapachone (2.5 mg/kg, intraperitoneally) could significantly attenuate the increase of serum TNF-{alpha} levels induced by LPS (Figure 7B).



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  		Figure 5. Effects of ß-lapachone (ß-LP) on LPS-induced lung edema and iNOS protein expression in mice. Mice were given vehicle (DMSO) or ß-lapachone (1.25, 2.5, or 5 mg/kg, intraperitoneally) simultaneously (simult.) with, or 1 hour after, the treatment of LPS (7.5 mg/kg, intraperitoneally). (A) Lung edema was measured as a wet/dry weight ratio in the mice that treated with LPS for 6 hours. Data are presented as means ± SEM of three experiments (three mice per group in each experiment). (B) Lung samples were prepared and subjected to Western blot analysis for iNOS as described in METHODS. {alpha}-Tubulin served as control for sample loading and integrity. Quantification of iNOS protein expression was performed by densitometric analysis. *p < 0.05 as compared with control (vehicle DMSO). #p < 0.05 as compared with LPS alone.

 


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  		Figure 6. Effect of ß-lapachone on LPS-induced mortality in mice. Mice were treated intraperitoneally with vehicle (DMSO; filled squares), LPS (25 mg/kg; open circles), ß-lapachone (ß-LP, 2.5 mg/kg; simultaneously with LPS; ß-LP + LPS, filled triangles), or 1 hour after LPS (LPS + ß-LP, open triangles), or ß-lapachone alone (filled circles). The mortality was monitored twice daily for 96 hours (10 mice per group).

 


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  		Figure 7. In vivo effects of ß-lapachone on TNF-{alpha} and nitrite production induced by LPS. (A) Mice treated intraperitoneally with LPS (7.5 mg/kg) for 6 hours to detect the changes of plasma nitrite levels. ß-Lapachone (ß-LP, 2.5 mg/kg, intraperitoneally) was administered simultaneously (simult.) with, or 1 hour after, LPS treatment. (B) Mice treated intraperitoneally with LPS (7.5 mg/kg) for 1 and 2 hours to detect the changes of serum TNF-{alpha} levels. ß-Lapachone (ß-LP, 2.5 mg/kg) and LPS were administered simultaneously. Data are presented as means ± SEM of three experiments (three mice per group in each experiment). *p < 0.05 as compared with control (vehicle-DMSO). #p < 0.05 as compared with LPS alone.

 
Furthermore, we performed experiments to test whether ß-lapachone could prevent the activation of NF-{kappa}B and ERK1/2 phosphorylation in lungs of mice after the administration of LPS in vivo. Mice were given vehicle (DMSO) or ß-lapachone (2.5 mg/kg, intraperitoneally) simultaneously with, or 1 hour after, the treatment of LPS (7.5 mg/kg, intraperitoneally). We observed an induction of ERK1/2 phosphorylation and an I{kappa}B{alpha} degradation in lungs of mice treated with LPS, which was prevented in the presence of ß-lapachone (Figure 8A) . To investigate further whether the ß-lapachone was capable of inhibiting the activation of NF-{kappa}B, the levels of NF-{kappa}B p65 protein in the nuclei of lungs were investigated with immunoblotting. The result showed an increase of NF-{kappa}B p65 protein expression in the nuclei of lungs during endotoxemia. ß-Lapachone effectively abolished this LPS-triggered NF-{kappa}B nuclear translocation in lungs (Figure 8B).



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  		Figure 8. Effects of ß-lapachone (ß-LP) on LPS-induced I{kappa}B{alpha} degradation, ERK1/2 phosphorylation, and NF-{kappa}B p65 protein translocation in lungs of mice. Mice were given vehicle (DMSO) or ß-lapachone (2.5 mg/kg, intraperitoneally) simultaneously (simult.) with, or 1 hour after, the treatment of LPS (7.5 mg/kg, intraperitoneally). Total cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with antibodies against I{kappa}B{alpha} protein and ERK1/2 phosphorylation. {alpha}-Tubulin served as control for sample loading and integrity. The nuclear extracts were prepared for analysis of NF-{kappa}B p65 protein translocation by Western blot as described in METHODS. Results shown are representative of at least three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ß-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho-[1,2-b]pyran-5,6-dione) is synthesized by simple sulfuric acid treatment of a natural plant product "lapachol," which is readily extracted from Tabecuia avellanedae, grown mainly in Brazil, or is easily synthesized from lomatiol, which is isolated from seeds of lomatia grown in Australia (1, 2). The major findings of this study are that ß-lapachone suppresses iNOS and TNF-{alpha} production mediated by the inhibition of protein tyrosine phosphorylation and NF-{kappa}B activation caused by LPS and protects mice from LPS-induced lung edema and lethal toxicity.

The murine macrophage iNOS is immunologically induced at the transcriptional level (16, 17). Activation of NF-{kappa}B is necessary for LPS induction of the iNOS gene expression (17, 23). In vivo inhibition of NF-{kappa}B activation has been found to prevent iNOS expression and systemic hypotension in a rat model of septic shock (23). In its unstimulated form, NF-{kappa}B is present in the cytosol bound to the inhibitory protein, I{kappa}B. Through noncovalent association, the I{kappa}B proteins mask the nuclear localization signal of NF-{kappa}B, thereby preventing NF-{kappa}B nuclear translocation. Activation of NF-{kappa}B is mediated through the phosphorylation of I{kappa}B, followed by the subsequent degradation of I{kappa}B at the proteasome. The study of Manna and colleagues (24) has shown that ß-lapachone could inhibit NF-{kappa}B activation in response to TNF-{alpha} and LPS in U937 cells. We have previously found that ß-lapachone was capable of inhibiting the LPS-induced expression of mRNA for iNOS in rat alveolar macrophages (8). To investigate the role of NF-{kappa}B in the effect of ß-lapachone on iNOS induction, we examined the NF-{kappa}B activation and I{kappa}B{alpha} degradation caused by LPS. The results indicate that ß-lapachone blocks the activation of NF-{kappa}B by inhibiting the removal of I{kappa}B{alpha}, the inhibitor, from the NF-{kappa}B/I{kappa}B{alpha} complex.

During gram-negative sepsis, human monocytes are triggered to produce large quantities of proinflammatory cytokines such as TNF-{alpha} in response to endotoxin. The regulation of monocyte function and the inhibition of TNF-{alpha} production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia (25). TNF-{alpha} transcription in macrophages is regulated by NF-{kappa}B (26, 28). NF-{kappa}B is activated in alveolar macrophages from patients with adult respiratory distress syndrome, and it is essential for the transcription of many cytokine genes including TNF (27). A recent study has also shown that the inhibition of NF-{kappa}B activation by pyrrolidine dithiocarbamate could prevent in vivo expression of TNF-{alpha} and other proinflammatory genes in response to LPS (28). Therefore, we examined the effect of ß-lapachone on the LPS-induced TNF-{alpha} production in alveolar macrophages and found that ß-lapachone was capable of inhibiting the TNF-{alpha} production in response to LPS, and it may be related to the suppression of NF-{kappa}B activation.

Recent reports have identified that LPS-induced NF-{kappa}B activation and cytokines released in human alveolar macrophages (29) and monocytes (22) were protein tyrosine kinase dependent. LPS- and cytokines-induced NO production initiates a common signaling pathway involving a protein tyrosine kinase that precedes the induction of NOS in murine macrophages (30) and in isolated vessels (31). It has also been reported that tyrosine kinase inhibitors of the tyrphostin AG126 family were capable of preventing LPS-induced lethal toxicity in mice (32) and improving survival and multiorgan failure in canine E. coli peritonitis (33). A recent study has indicated that the src family-selective tyrosine kinase inhibitor pyrazolopyrimidine PP1 could block LPS and interferon-{gamma}–mediated TNF and iNOS production in murine macrophages (34). Moreover, LPS induces protein tyrosine phosphorylation of a handful of proteins; mitogen-activated protein kinases are several of the more prominent phosphorylated proteins. Stimulation of macrophages by LPS leads to the rapid activation of mitogen-activated protein kinases and the subsequent induction of cytokine gene expression. PD98059, an inhibitor of the ERK pathway, has been shown to block LPS-induced activation of TNF-{alpha} gene expression in an alveolar macrophage cell line (35). A recent study has also shown that TNF-{alpha} induced by LPS in murine macrophages is regulated post-transcriptionally via a Tp12/ERK-dependent pathway (36). Therefore, we investigated the effect of ß-lapachone on the protein tyrosine phosphorylation. Our results indicated that LPS-stimulated protein tyrosine phosphorylation, including mitogen-activated protein kinase family ERK1/2, was significantly inhibited by ß-lapachone in rat alveolar macrophages. These findings imply that the suppression of LPS-induced responses in macrophages by ß-lapachone is related to protein tyrosine kinase and NF-{kappa}B activation.

Acute lung injury has been observed in animals and patients during bacterial sepsis (38, 39). NO synthase inhibitors could prevent this LPS-induced acute lung injury (39, 40). LPS has been shown to lead to large amounts of NO in alveolar macrophages, lung epithelial cells, and endothelial and interstitial cells (41). The NO generated by iNOS is thought to have a key role in LPS-induced acute lung injury in animal models. Kobayashi and colleagues (42) have suggested that iNOS together with proinflammatory cytokines produced by alveolar macrophages might play a pivotal role in the pathogenesis of acute lung injury in adult respiratory distress syndrome patients after sepsis. Moreover, TNF is considered to be a major early mediator in the systemic inflammatory response syndrome observed during Gram-negative sepsis (43). It has been shown that inhibition of TNF dramatically protected mice from the lethal effect of endotoxin (44). Recently, Reinhart and Karzai (45) have suggested that the analysis of all trial data, as well as data from a recent trial in a large population of septic patients, shows that anti-TNF strategies may confer a small survival benefit, although individual studies show small, nonsignificant benefits. In this work, the results of in vivo studies showed that ß-lapachone was capable of reducing the LPS-induced lung edema and lethal toxicity in mice when it was administrated simultaneously with, or 1 hour after, LPS treatment. Moreover, we also found that ß-lapachone could prevent the activation of NF-{kappa}B and ERK phosphorylation in lungs of mice after the administration of LPS in vivo. The increased serum TNF-{alpha} and total plasma nitrite levels in LPS-treated mice were also suppressed by ß-lapachone. Thus, it seems possible that these protective effects of ß-lapachone may correlate with its inhibition of LPS-induced production of NO and TNF-{alpha} in macrophages and may be effective in preventing the septic shock in gram-negative infections. Nevertheless, in our in vivo experimental condition, production of TNF-{alpha} peaks early (approximately 1 hour; Figure 7B) in endotoxemia and then declines back to control levels after 3 to 4 hours (data not shown) of LPS injection, but the influence of endotoxemia in lung edema and lethality was displayed at 6–96 hours after LPS injection. Moreover, ß-lapachone was still effective in reducing the LPS-induced mortality when it was given 1 hour after the application of LPS. In a LPS-based in vivo model, Heremans and colleagues (39) have shown that NO production contributed to the development of early lung edema, but interferon-{gamma} might contribute to later lethal events. Therefore, in the future, further studies are needed to clarify the role of TNF-{alpha} and/or other cytokines such as interferon-{gamma} in LPS-induced lethal toxicity and the effects of ß-lapachone on these responses.

In summary, in this work, the primary cell culture of rat alveolar macrophages and mouse macrophage cell line RAW264.7 were used for in vitro studies, and a LPS-treated BALB/c mouse model, which exhibits pathogenic similarities with human septic shock conditions (46), was used for in vivo studies. We performed experiments to examine the intracellular mechanism of ß-lapachone on LPS-induced iNOS and TNF-{alpha} production in alveolar macrophages and in vivo LPS-induced lung edema, ERK1/2 phosphorylation, NF-{kappa}B activation, lethal toxicity, and increased NO and TNF-{alpha}. We found that ß-lapachone was capable of suppressing LPS-induced iNOS and TNF-{alpha} production by inhibition of protein tyrosine phosphorylation and NF-{kappa}B activation and exerts a beneficial effect in an animal model of sepsis.


   FOOTNOTES
 
Supported by grants from the National Science Council of the Republic of China (NSC85-2331-B-002-292 and 89-2320-B-002-179).

Received in original form September 16, 2002; accepted in final form April 25, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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