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ABSTRACT |
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INTRODUCTION
METHODS
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Luteolin is a flavonoid that has been shown to reduce proinflammatory molecule expression in vitro. In the present study, we have
tested the ability of luteolin to inhibit lipopolysaccharide (LPS)-
induced lethal toxicity and proinflammatory molecule expression in vivo. Mice receiving LPS (Salmonella enteriditis LPS, 32 mg/kg, intraperitoneally) exhibited high mortality with only 4.1% of the animals surviving seven days after the LPS challenge. On the contrary,
mice that had received luteolin (0.2 mg/kg, intraperitoneally) before LPS showed an increased survival rate with 48% remaining alive on Day 7. To investigate the mechanism by which luteolin affords protection against LPS toxicity we measured intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-
(TNF-) production in response to LPS in the presence or absence of luteolin pretreatment. Treatment of animals with LPS increased serum
TNF- levels in a time-dependent manner. The increase in peak serum TNF- levels was sensitive to luteolin pretreatment. Luteolin pretreatment also reduced LPS-stimulated ICAM-1 expression in the liver and abolished leukocyte infiltration in the liver and lung.
We conclude that luteolin protects against LPS-induced lethal toxicity, possibly by inhibiting proinflammatory molecule (TNF-, ICAM-1) expression in vivo and reducing leukocyte infiltration in tissues.
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INTRODUCTION |
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Keywords: luteolin; tumor necrosis factor-; intercellular adhesion
molecule; lipopolysaccharide; sepsis
Septic shock affects more than 300,000 patients worldwide and
has a high mortality of up to 60% despite antibiotic therapy and intensive care support (1, 2). The potentially fatal syndrome
of irreversible cardiovascular collapse and critical organ failure was originally attributed to lipopolysaccharide (LPS), an
outer membrane component of gram-negative bacteria. However, it is the endogenously produced proinflammatory molecules, rather than LPS itself, that mediate the pathophysiological alterations that accompany endotoxemia; endotoxin acts on
many different cell types stimulating the expression of cytokines and adhesion molecules that contribute to the inflammatory response (3-5). One cytokine believed to play a major
role in sepsis is tumor necrosis factor- (TNF-) (6). Serum
levels of TNF- reach nanomolar concentrations in experimental models of sepsis and administration of human recombinant TNF- to animals induces circulatory collapse and
critical organ injury that is similar to that seen in lethal endotoxemia (7). Moreover, an anti-TNF- monoclonal antibody
protects baboons from shock caused by bacteremia and homozygous TNF- mutant mice, for both p55 and p75 are resistant to sepsis (8, 9).
Intercellular adhesion molecule-1 (ICAM-1) is a member
of the immunoglobulin superfamily expressed on endothelial
cells. ICAM-1 is important for adhesion of leukocytes and
transendothelial migration through binding to the 
2 integrins
present on leukocytes LFA-1 and MAC-1 (10). Exposure of
endothelial cells to inflammatory stimuli leads to increased expression of ICAM-1, facilitating leukocyte accumulation in tissues (11, 12). The importance of ICAM-1 in sepsis is underscored by the observation that mice with targeted disruption
of the ICAM-1 gene locus show reduced infiltration of neutrophils to the liver and are resistant to the lethal effects of high
doses of LPS, in spite of similar increases in circulating levels
of cytokines (13, 14).
Flavonoids are naturally occurring polyphenolic compounds
with a wide distribution in the plant kingdom. It is thought
that the average western diet contains approximately 1 g of
flavonoids per day (15). We and others (16-20) have shown
that flavonoids inhibit proinflammatory molecule expression
in vitro. Luteolin, a flavone found in high concentrations in
celery, green pepper, perilla leaf and seeds, and chamomile, is
among the most potent and efficacious flavonoid inhibitors of
LPS-induced TNF-, interleukin-6 production and inducible
nitric oxide expression (17). In the present study, we extend
our in vitro findings that luteolin blocks LPS-induced cytokine
release by demonstrating that luteolin also attenuates TNF-
production and ICAM-1 expression in vivo and abolishes infiltration of leukocytes in the lung and liver of LPS-treated mice.
More importantly, pretreatment with luteolin reduces the
mortality associated with LPS administration in a mouse
model of sepsis.
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METHODS
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Reagents and Cell Culture
Luteolin was obtained from Roth Chemicalien (Karlsruhe, Germany).
Luteolin was found to contain only trace amounts of endotoxin (25 ng
endotoxin/g luteolin) TNF- and interleukin (IL)-6 ELISA kits were
from R&D Systems (Minneapolis, MN). Tissue culture plates were
from Nalge Nunc International (Rochester, NY). Protein dye reagent
and the chemiluminesence Western blotting system were from Pierce
(Rockford, IL). Dulbecco's modified Eagle medium (DMEM), fetal
calf serum (FCS), and antibiotics were obtained from Gibco BRL
(Paisley, UK). The ICAM-1 antibody (H-108/sc-7891) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the secondary peroxidase-conjugated goat anti-rabbit antibody was from
Jackson ImmunoResearch Lab (West Grove, PA). All other reagents including LPS (Salmonella enteriditis) were obtained from Sigma Chemical (St. Louis, MO). RAW 264.7 cells were cultured in low glucose DMEM containing 10% fetal bovine serum supplemented with penicillin and streptomycin at 37° C in a humidified incubator with
5% CO2.
LPS Lethality Studies
C57BL/6 male and female mice 10-12 weeks of age were pretreated with vehicle or luteolin (0.2 mg/kg, intraperitoneally) for 30 minutes before the LPS challenge (Salmonella enteriditis 32 mg/kg intraperitoneally). Injections were made in a total volume of 100 µl. Animals were observed for seven days.
TNF- and IL-6 Measurements
Cytokine measurements in RAW 264.7 cells were performed as previously described (17). For serum TNF- measurements, mice (C57BL/6
10-12 weeks old) were pretreated with luteolin (0.2 or 1 mg/kg, intraperitoneally, for 30 or 120 minutes) or vehicle (dimethylsulfoxide,
DMSO) followed by LPS (32 mg/kg intraperitoneally). After the indicated time, blood samples were collected. They were then allowed to
clot for two hours at room temperature, centrifuged for 20 minutes at
2,000 × g, and stored at 
80° C until measured.
Western Blotting
Mice (C57BL/6 10-12 weeks old) were pretreated with luteolin (0.2 mg/ kg, intraperitoneally, for 30 minutes) or vehicle (DMSO) followed by LPS (32 mg/kg, intraperitoneally). After eight hours, animals were sacrificed and tissues removed and snap frozen in liquid nitrogen. Proteins were extracted from tissues as previously described with minor modifications (21). Equal amounts of proteins were loaded and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were blotted to nitrocellulose membrane, incubated with antibodies, and visualized using enhanced chemiluminescence Western blotting detection system.
Histopathological Studies
Mice (three or four per group) were treated with vehicle (DMSO), LPS (32 mg/kg, intraperitoneally), or LPS plus luteolin (0.2 mg/kg, intraperitoneally, 30 minutes pretreatment) and sacrificed 10 hours after LPS exposure by hypercapnia. Lungs and livers were removed immediately by thoracotomy and laparotomy. The right lungs were used for histological studies. The tissue specimens were fixed overnight in 4% buffered formaldehyde, processed by standard methods, and stained for hematoxylin and eosin (H&E). Semiquantitative analysis of tissues was performed by one observer in a blinded fashion.
Data Analysis and Statistics
Data are presented as means ± SEM of the indicated number of observations. For cytokine measurements statistical comparisons between groups were performed using the one-way ANOVA followed by the Dunnett's post hoc test. Differences among means were considered significant when p < 0.05. Survival as the end point in the lethality experiments was calculated from the time of treatment using the product limit Kaplan-Meier method. Differences in the Kaplan- Meier survival curves were evaluated by the Mantel log-rank test for censored data.
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RESULTS |
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INTRODUCTION
METHODS
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Effects of Luteolin on TNF- Production from
Murine Macrophages
We have previously shown that flavonoids inhibit TNF- production from RAW 264.7 cells in response to Escherichia coli
LPS (serotype 0B26) (17). Because for our in vivo studies we
used LPS derived from Salmonella enteriditis, we tested
whether luteolin can inhibit TNF- production from RAW
264.7, in response S. enteriditis LPS. Exposure of RAW 264.7 to S. enteriditis LPS led to a 6-fold increase in the release of
TNF- in the culture medium. Pretreatment of the macrophages with 10 µM luteolin for 30 minutes led to a significant
inhibition of the LPS-induced TNF- release (Figure 1).
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Effect of Luteolin on Survival in a Mouse Model of Sepsis
To test if luteolin can increase survival in a mouse model of sepsis, 10- to 12-week-old animals were injected with DMSO or luteolin (0.2 mg/kg) before LPS (32 mg/kg, intraperitoneally, S. enteriditis LPS). The dose of LPS used is close to the LD95 and the dose of luteolin chosen for the lethality studies was the one that showed the most promising results in pilot experiments. Mice receiving DMSO or luteolin alone exhibited no lethality, whereas only 4.1% of the mice receiving LPS survived after seven days (Figure 2). Pretreatment with luteolin increased survival rate throughout the time of observation with 48% of the mice pretreated with luteolin remaining alive after seven days. In contrast, a higher dose of luteolin (10 mg/ kg) offered no significant protection against LPS-induced lethal toxicity (none of six mice alive in the LPS group after seven days versus one of six in the luteolin + LPS group). Administration of a 50 mg/kg dose of luteolin in the absence of LPS was lethal to three of three mice receiving it.
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Vehicle;
LPS; 
Lut + LPS; 
Lut.
Effects of Luteolin on LPS-induced Proinflammatory Molecule Expression in Vivo
To determine the mechanism by which luteolin protects animals
from LPS lethal toxicity we measured its ability to lower serum TNF- levels in the context of sepsis. Dimethylsulfoxide
(vehicle)-treated mice had undetectable levels of circulating
TNF-. Pretreatment of mice with luteolin (0.2 or 1 mg/kg luteolin intraperitoneally) for 30 or 120 minutes before the LPS challenge attenuated the increase in serum TNF- levels elicited
by LPS (Figure 3). However, when luteolin (0.2 mg/kg, intraperitoneally) was given 30 minutes after the LPS, no reduction
in serum TNF- was observed (6.6 ± 1.0 pg/ml for the LPS
group versus 10.5 ± 1.4 pg/ml for the luteolin + LPS group;
n = 3). In addition, the 10 mg/kg dose of luteolin that failed to
protect animals from the lethal toxicity of LPS was effective in
reducing serum TNF- levels (0.9 ± 0.3 pg/ml). Injection of
mice with LPS increased serum TNF- levels in a time-dependent manner, reaching a peak after 60 to 90 minutes (Figure
4A). Luteolin significantly reduced peak TNF- levels, without affecting the early rise in TNF- levels at 30 minutes (Figure 4A). In addition, luteolin showed no overall effect in circulating IL-6 levels (Figure 4B).
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We next investigated the ability of luteolin to affect ICAM-1 expression, an important regulator of leukocyte trafficking. Western blot analysis showed that ICAM-1 protein expression was markedly up-regulated by the LPS challenge in liver homogenates after eight hours. Pretreatment of animals with 0.2 mg/ kg luteolin prevented the up-regulation of the protein (Figure 5).
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Effects of Luteolin on Leukocyte Accumulation in the Lung and Liver
To test the functional relevance of the reduced ICAM-1 expression in the face of luteolin pretreatment, we studied the inflammatory infiltration in organs that constitute major targets of LPS. Histopathologic analysis of H&E-stained tissue sections from LPS-treated mice revealed a marked peribronchiolar and perivascular infiltration of leukocytes in the lung and around portal or central spaces in the liver (Figures 6B and 6E). Pretreatment with luteolin abolished the accumulation of leukocytes in both organs (Figures 6C and 6F and Table 1). However, luteolin did not prevent more subtle changes in tissue architecture caused by LPS, such as the thickening of alveolar septa and the hyperplasia of bronchial epithelium in the lung or the hyperplasia of vascular endothelium in the liver, as well as hyperplasia of the epithelium in the bileary ducts (Figures 6C and 6F).
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DISCUSSION |
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In sepsis, challenge of cells with LPS stimulates cascades leading to phosphorylation and activation of multiple mitogen-activated protein kinase family members (Erk1, Erk2, p46/p54 JNK/ SAPK, and p38), as well as phosphoinositide-3 kinase/Akt and nonreceptor tyrosine kinases of the Src family (Lyn, Fgr, and Hck) (22-26). Blocking LPS-stimulated phosphorylation cascades has been proposed as a promising target for the development of novel therapeutic agents for patients with sepsis (27). For example, tyrphostins, a class of agents with tyrosine kinase blocking activity, increase survival of mice injected with LPS (28).
The major finding of the present study is that the flavonoid luteolin protects mice from LPS-induced lethal toxicity. The increased survival of mice pretreated with luteolin is not entirely surprising as flavonoids are known inhibitors of serine/threonine and tyrosine protein kinases (29). Indeed, flavonoids have been described to act as competitive inhibitors with respect to the ATP
binding site on a variety of kinases, a region of considerable homology among the kinase family (30, 31). This property enables flavonoids to block the activity of both lipid and protein kinases, such as PI3-K and PKC (32). Inhibition of a broad spectrum of kinases could be advantageous as it could interfere with many cascades involved in sepsis. Moreover, in addition to inhibiting phosphorylation events, flavonoids block the activity of a variety of enzymes involved in inflammation, including lipo- and cyclooxygenases (29, 33, 34). We have previously shown that luteolin blocks LPS-stimulated tyrosine phosphorylation and Akt activation in cultures of the macrophage cell line RAW 264.7, leading to inhibition of NF-
B activation (17), a transcription factor known to mediate expression of many inflammatory genes
(35). Recently, Takahashi and coworkers (36) compared a series
of flavonoids (quercetin, rutin, baicalein, baicalin, daidzein, catechin, and hesperetin) in two models of sepsis. Pretreatment of
mice with the flavonoids (4 mg/kg) protected against the lethal
response to a high dose of LPS, as well as the lethal response that
follows LPS injection in D-galactosamine-sensitized mice. These
findings are in line with our observations on the protective effects
of luteolin and strengthen our hypothesis that flavonoids can be
used to block the cascades triggered by endotoxin in vivo.
Most of the studies on luteolin's actions have been performed in vitro (isolated organ or cell culture) (17, 20, 37, 38).
In vivo, luteolin has been reported to exhibit antifertility, antiallergic, and radioprotective properties (39-41). To investigate the mechanisms through which luteolin reduces LPS-induced
lethal toxicity we determined the ability of luteolin to decrease serum TNF- levels in mice challenged with LPS.
Treatment of animals with LPS resulted in a time-dependent
increase in circulating TNF- that peaked at 60 to 90 minutes.
Pretreatment of mice with luteolin (0.2-1 mg/kg) reduced
peak TNF- production, whereas administration of luteolin
after the LPS did not inhibit TNF- levels. Interestingly, pretreatment of mice with a higher dose of luteolin (10 mg/kg),
which failed to protect from the lethal toxicity of LPS, decreased
serum levels of TNF-, suggesting that at high concentrations
luteolin, in addition to inhibiting inflammatory cytokine production, inhibits pathways that offer protection against LPS lethal toxicity. Rutin, another flavonoid, reduced the ability of
LPS to increase serum TNF- levels in D-galactosamine-sensitized mice, but was ineffective in reducing serum TNF- in
mice receiving a high dose of LPS (36).
Unlike serum TNF-, luteolin did not reduce circulating
IL-6 in LPS-treated animals. This is in contrast to our observation that luteolin inhibits LPS-induced IL-6 production from
RAW 264.7 and that apigenin, a related flavone, inhibits IL-6
release from endothelial cells (17, 19). IL-6 is produced by a
variety of cells in vivo in response to LPS, but the relative contribution of each cell type remains unclear. Although there is no
evidence showing that luteolin selectively inhibits IL-6 production in some cell types, it is possible that the inability of luteolin to block IL-6 release results from a lack of effect of luteolin on
certain IL-6-producing cells. Alternately, luteolin may not be
distributed in compartments in which most of the IL-6 production occurs.
Expression of leukocyte-specific adhesion molecules on the
endothelial cell surface is induced by LPS or inflammatory cytokines. In vivo, during sepsis, up-regulation of leukocyte adhesion molecules promotes leukocyte accumulation in tissues,
which enhances inflammation and contributes to the multiple
organ dysfunction syndrome (13, 14). Inhibiting the expression
of these molecules represents a major target for antiinflammatory therapy. One molecule known to be important for leukocyte trafficking through endothelial monolayers is ICAM-1
(10). Flavonoids, such as apigenin and luteolin, reduce ICAM-1,
vascular cell adhesion molecule-1, and E-selectin expression
induced by TNF- and interferon-
treatment of human endothelial cells (19). Apigenin inhibits neutrophil and peripheral blood lymphocyte adhesion to cytokine-stimulated EC
monolayers and blocks TNF--induced ICAM-1 expression in
vivo (12). To test if luteolin is capable of blocking ICAM-1 expression in the context of sepsis, we determined ICAM-1 expression in the liver of LPS-treated mice with or without luteolin pretreatment. The increase in ICAM-1 expression due
to LPS was abolished by luteolin. Moreover, leukocyte infiltration in the lung and liver was reduced in the face of luteolin
pretreatment, providing evidence that reduced expression of
ICAM-1 in luteolin-treated mice correlates functionally with a
decreased leukocyte migratory response.
In summary, we have demonstrated that the flavonoid luteolin can protect mice from LPS-induced lethal toxicity. Moreover, luteolin inhibits LPS-induced TNF- production, ICAM-1
expression, and leukocyte accumulation in tissues. More extensive screen of this class of compounds and preparation of
synthetic analogues might yield molecules with increased potency and efficacy that could be useful for patients with sepsis.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Andreas Papapetropoulos, Ph.D.,"George P. Livanos" Laboratory, University of Athens, Ploutarchou 3, Athens, Greece 10675. E-mail: papapetropoulos{at}yahoo.com
(Received in original form January 11, 2001 and accepted in revised form November 26, 2001).
Acknowledgments: The authors wish to thank Dr. George Kollias for providing the C57BL/6 mice and Dr. Dimitris Kontogiannis for his help with the in vivo experiments.
Supported by a grant from the Greek Secretariat of Research and Technology and the Thorax Foundation.
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