Third Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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ABSTRACT |
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Natural immunomodulator
lactoferrin is known to exert an anti-inflammatory effect. However,
there have been no studies that examine the mode of action of
lactoferrin in reducing intestinal damage. We investigated the effect
of lactoferrin on a trinitrobenzenesulfonic acid (TNBS)-induced colitis
model in rats. Bovine lactoferrin was given once daily through gavage,
starting 3 days before (preventive mode) or just after TNBS
administration (treatment mode) until death. The distal colon was
removed to be examined. Colitis was attenuated by lactoferrin via both
modes in a dose-dependent manner, as reflected by improvement in
macroscopic and histological scores and myeloperoxidase activity.
Lactoferrin caused significant induction of the anti-inflammatory
cytokines interleukin (IL)-4 and IL-10, significant reductions in the
proinflammatory cytokines tumor necrosis factor- and IL-1
, and
downregulation of the nuclear factor-
B pathway. We concluded that
lactoferrin exerts a protective effect against colitis in rats via
modulation of the immune system and correction of cytokine imbalance.
Lactoferrin has potential as a new therapeutic agent for inflammatory
bowel disease.
trinitrobenzenesulfonic acid; inflammatory bowel disease; tumor
necrosis factor; interleukin; nuclear factor-B
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INTRODUCTION |
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THE ETIOLOGY OF
CROHN'S DISEASE and ulcerative colitis is still unknown.
However, genetic factors in combination with environmental factors are
suggested to be involved in the pathogenesis of Crohn's disease
(31). Inadequate or prolonged activation of the intestinal immune system plays an important role in the pathophysiology of chronic
mucosal inflammation (11, 24, 31). Furthermore, cytokines
play an important role in modulating the immune system. Cytokines are
rapidly synthesized and secreted from inflammatory cells on stimulation
and induce the production of adhesion molecules and other inflammatory
mediators such as reactive oxygen metabolites, nitric oxide, and lipid
mediators (1). Cytokines induce, amplify, prolong, and
inhibit inflammation (3, 29). It has been reported that
there is a disturbed balance between proinflammatory and anti-inflammatory cytokines in inflammatory bowel disease (3, 24,
29). Increased levels of proinflammatory cytokines such as tumor
necrosis factor- (TNF-
), interleukin (IL)-1
, and IL-6 were
detected in the colonic mucosa. They are secreted from macrophages, lymphocytes, and polymorphonuclear neutrophils (13, 21,
32). Synthesis of these cytokines is induced by the activation
of nuclear factor (NF)-
B, a transcriptional factor that is involved
in the regulation of many inflammation-associated genes
(4).
On the basis of these observations, we speculated that appropriate
modulation of the immune system by using natural immunomodulators might
be useful in reducing the activity of inflammatory bowel disease. We
focused our attention to lactoferrin, an iron-binding protein with a
wide spectrum of biological activities. Lactoferrin exists in various
biological fluids such as milk, tears, synovial fluid, seminal plasma,
and pancreatic juice (22). Lactoferrin is stored in
secondary granules of neutrophils and is released in response to
activation of TNF-, IL-6, and IL-8 (7). The serum level
of lactoferrin, which is very low in the physiological state, increases
significantly with infection (22). Receptors for
lactoferrin were detected and isolated in activated B and T
lymphocytes, monocytes/macrophages, intestinal brush-border cells,
platelets, and neoplastic cells (22). Lactoferrin also influences the release of proinflammatory cytokines such as IL-1
, IL-2, IL-6, and TNF-
(7, 16, 18, 25, 37).
In this study, we examined the potential ability of lactoferrin to
attenuate colitis using a 2,4,6-trinitrobenzenesulfonic acid
(TNBS)-induced colitis model in rats. TNBS has been shown to induce
colitis rapidly and reproducibly as a result of covalent binding of
trinitrophenyl residues to autologous host proteins, leading to mucosal
infiltration by polymorphonuclear cells (10), activation of the NF-B pathway (27), and the production
of TNF-
and IL-1
(28).
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MATERIALS AND METHODS |
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Animals
Male Sprague-Dawley rats (7 wk of age, weighing 210-240 g) were obtained from Clea Japan (Tokyo, Japan) and used in the experiments. Rats were housed in hanging wire mesh cages in a temperature- (23 ± 2°C) and humidity- (50-70%) controlled room with a 12:12-h light-dark cycle. Animals were fed ad libitum with standard rat chow (Clea Japan) and had free access to tap water throughout the experimental period. The rats were allowed to adapt to our laboratory environment for 1 wk before beginning the experiment. This animal study was approved by the Ethics Committee of Yokohama City University.Induction of Colitis
Colonic inflammation was induced by using a technique modified from that of Morris et al. (26). Briefly, rats were lightly anesthetized with ether after overnight food deprivation, and a polyethylene catheter was inserted 8 cm into the colon via the anus. TNBS (Wako Chemical, Tokyo, Japan) dissolved in 50% (vol/vol) aqueous ethanol (25 mg/ml) was injected into the colon (total volume of 1 ml/rat). The control rats received 1 ml of water intracolonically in place of TNBS and ethanol.Experimental Procedures
Experiment 1: protective effects of lactoferrin on TNBS-induced
colitis according to mode of administration.
To evaluate the preventive effect of lactoferrin against
TNBS-induced colitis, rats were divided into three groups:
1) TNBS-administered rats receiving 0.9% saline,
2) TNBS-administered rats receiving lactoferrin, and
3) nontreated controls (n = 8 in each
group). Bovine lactoferrin (200 mg · kg1 · day
1;
Morinaga Milk Industry, Tokyo, Japan) dissolved in 1 ml of 0.9% saline was administered once a day through gavage, starting from 3 days
before colitis induction and continuing until death (preventive mode).
The dose of lactoferrin was selected on the basis of the report that no
adverse effect in rats was observed at the level of lactoferrin between
0 and 2,000 mg · kg
1 · day
1
(40). Animals were weighed every day until death.
Macroscopic and histological examinations, as well as determination of
myeloperoxidase (MPO) activity, in the distal colon were conducted
before, 7, and 14 days after TNBS administration.
Experiment 2: dose-response study of lactoferrin in TNBS-induced
colitis.
Dose-response relationships between lactoferrin and the degree of
inflammation scored macroscopically and histologically and by measuring
MPO activity in the distal colon were examined in four groups of
animals 7 days after TNBS administration. Different doses of
bovine lactoferrin (0, 50, 100, and 200 mg · kg1 · day
1) were
administered once a day from 3 days before colitis induction until death.
Experiment 3: effects of lactoferrin on cytokines in the colonic
tissue and the NF-B pathway in TNBS-induced colitis.
In other groups of TNBS-administered rats and control rats
(n = 8 in each group) receiving lactoferrin in the
preventive mode, the distal colon was resected. Proinflammatory
cytokines, anti-inflammatory cytokines, and NF-
B were determined at
7 days after TNBS administration.
Macroscopic Assessment of Colonic Inflammation
The distal colon was quickly removed from the rats, opened longitudinally, and gently cleared of fecal material with the 0.9% saline solution. Colonic inflammation was evaluated macroscopically with the colon macroscopic scoring system of Wallace et al. (39) by an observer unaware of the treatment. This score is scaled from 0 to 10 based on criteria reflecting inflammation, such as hyperemia, bowel wall thickening, and extent of ulceration. After being scored, the distal 10 cm of the colon was blotted dry and weighed. The colon weight-to-body weight ratio was calculated as a marker of colonic inflammation (34).Histological Assessment of Colonic Inflammation
Samples of the inflamed tissues were removed for histology. The colonic inflammation score was evaluated by using the histopathological grading system of Ameho et al. (2) by an observer blinded to the treatment. This grading, which takes into account the degree of infiltration, the presence of erosion, ulceration, or necrosis, and the depth and surface of the lesion, is scaled from 0 to 6.Determination of the Plasma and Fecal Concentrations of Lactoferrin
Plasma concentration of lactoferrin was measured by an ELISA assay kit (Yagai, Yamagata, Japan) according to the manufacturer's instructions. Fecal samples obtained from rectum were weighed, homogenized in buffer solution, and centrifuged, and lactoferrin in the supernatants was measured by ELISA by using the same kits.Determination of the Total Number of Colonic Bacteria
Fresh fecal samples were collected from rectum, and the bacterial numbers in feces were assayed as described previously (17).Determination of MPO Activity in the Colonic Tissue
Measurement of MPO activity is used as a biochemical marker of neutrophil infiltration into inflamed intestinal tissue (10, 26, 39). The MPO activity of the tissue was determined by the technique described by Bradley et al. (6).Determination of Cytokine Concentrations in the Colonic Tissue
Samples of inflamed colon were weighed and homogenized for 1 min in 10 mM phosphate buffered saline, pH 7.4, containing 1 mM EDTA, 5 mM dithiothreitol, and 10 µg/ml of each of aprotinin, leupeptin, and pepstatin A in an ice-chilled water bath, using a polytron homogenizer. The specimens were freeze-thawed four times. The homogenates were centrifuged at 10,000 g at 4°C for 5 min. TNF-Detection of Phosphorylated Inhibitor B in Cytoplasm
Statistical Analysis
All data are presented as means ± SE. Differences between groups were determined by analysis of variance followed by Fisher's protected least significant difference comparison test. With all statistical analyses, an associated probability (P value) of <0.05 was considered significant. ![]() |
RESULTS |
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Experiment 1: Effects of Preventive vs. Treatment Mode of Lactoferrin Administration on TNBS-Induced Colitis
Body weight gain was significantly suppressed in the TNBS-administered rats in contrast to the control rats (P < 0.01) in the first 2 days. TNBS-administered rats exhibited severe watery diarrhea and hemorrhaging over the following days. Administration of lactoferrin (preventive mode) attenuated the TNBS inhibition on body weight (P < 0.01 vs. TNBS-only rats) in the first 2 days. In the following days, there was no difference in body weight gain among the three groups (Fig. 1).
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Control rats killed 7 or 14 days after administration of saline showed
no macroscopic lesions in the distal colon (Fig.
2A). In the TNBS-administered
rats, very severe colitis with widespread hemorrhagic damage in the
distal colon was observed 7 days after TNBS administration (Fig.
2B). Fourteen days after colitis induction, the lesions were
still severe, although a tendency for improvement was observed. The
macroscopic inflammation score was the highest at 7 days after TNBS
administration and decreased subsequently, although it remained higher
than in control rats until 14 days after administration (Table
1). Lactoferrin treatment in the prevention mode attenuated the macroscopic lesions observed 7 and 14 days after TNBS administration (Fig. 2C). Seven and fourteen days after colitis induction, a significant decrease in the macroscopic score (P < 0.01) was observed in rats that had
received preventive lactoferrin compared with TNBS rats.
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No histological abnormalities were detected in the control rats killed
7 and 14 days after saline administration (Fig.
3A). In TNBS rats, colonic
inflammation extending deeply into the muscular layer was observed 7 days after administration. A polymorphic inflammatory infiltrate, rich
in polynuclear neutrophils, and local ulceration of the mucosal layer
were observed (Fig. 3B). Fourteen days after colitis
induction, the lesions were still severe, with marked infiltration of
neutrophils, and a tendency for improvement was observed. The
histological inflammation score increased to a peak level at 7 days
after TNBS administration and remained higher than in controls until 14 days after administration (Table 1). Lactoferrin dramatically reduced
the inflammatory lesions, which consisted of smaller polymorphic
infiltrates, limited edema, and small focal lesions indicative of
repair of the mucosal layer (Fig. 3C). A significant
decrease in histological score (P < 0.01) was observed
in TNBS-administered rats given preventive lactoferrin compared with
those given only TNBS.
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The colon weight-to-body weight ratio, an index of colonic inflammation, also increased after TNBS administration, reached its peak level on day 7, and decreased subsequently (Table 1). Preventive lactoferrin significantly reduced the colon weight-to-body weight ratio (P < 0.01).
MPO activity in the colonic tissue was significantly higher in TNBS-administered rats compared with controls at 7 or 14 days after TNBS administration (Table 1). Lactoferrin administration caused a significant decrease in MPO activity compared with TNBS-administered rats at 7 and 14 days after TNBS administration (P < 0.01), consistent with decreased infiltration of neutrophils, which was observed in histology.
Table 2 shows the effects of the
preventive and treatment modes of lactoferrin administration on
TNBS-induced colitis. Administration of lactoferrin in the treatment
mode also significantly reduced the macroscopic and histological
inflammation scores, the colon weight-to-body weight ratio, and
MPO activity at 7 days after TNBS administration. There were no
significant differences in these parameters between the
TNBS-administered rats in the treatment mode and those in the
preventive mode of lactoferrin administration.
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In TNBS-administered rats receiving lactoferrin, the plasma and fecal concentrations of lactoferrin 7 days after colitis induction were significantly increased (plasma concentration, 225 ± 40 ng/ml; fecal concentration, 12.2 ± 1.6 µg/g of feces). In control rats and TNBS-administered rats without lactoferrin, neither plasma nor fecal concentrations of lactoferrin were detectable.
With regard to the total bacterial numbers in feces 7 days after colitis induction, there were no significant differences between TNBS-administered rats receiving lactoferrin, TNBS-administered rats without lactoferrin, and control rats (9.28 ± 0.10, 9.39 ± 0.12, and 9.36 ± 0.15, respectively; data expressed as log10 colony-forming units/g feces).
Experiment 2: Dose-Response Study of Lactoferrin in TNBS-Induced Colitis
Macroscopic and histological damage scores, the colon weight-to-body weight ratio, and tissue levels of MPO activity in TNBS-administered rats were reduced by lactoferrin treatment in a dose-dependent manner within 50-200 mg · kg
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Experiment 3: Effects of Lactoferrin Administration on Cytokines in
the Colonic Tissue and NF-B Pathway in TNBS-Induced Colitis
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Figure 6 shows immunohistochemical
localization of phosphorylated IB, an index of NF-
B pathway
activation, in TNBS-induced colitis 7 days after colitis induction.
Phosphorylated I
B was induced in rats 7 days after TNBS
administration (Fig. 6B) compared with controls, which
showed almost no staining (Fig. 6A). Administration of
lactoferrin in the preventive mode attenuated phosphorylated I
B
expression (Fig. 6C).
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DISCUSSION |
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TNBS-induced colitis is a well-established model that is similar
to human inflammatory bowel disease and is characterized by mucosal
infiltration of neutrophils mediated, at least in part, by TNF- and
IL-1
activation (27) and activation of the NF-
B pathway (28). There are a number of studies that have
shown that hapten reagent-induced colitis, induced by TNBS, favorably responds to some of the current therapies for inflammatory bowel disease such as sulfasalazine, glucocorticoids, cyclosporine
(5), and anti-TNF-
antibodies (27). In
this study, we examined the effects of lactoferrin on TNBS-induced
colitis in Sprague-Dawley rats. Since it was reported that the
lactoferrin showed anti-inflammatory and immunomodulatory effects
(7, 22, 25, 37), lactoferrin is expected to attenuate
exaggerated activation of proinflammatory cytokines in TNBS colitis.
Oral administration of lactoferrin in TNBS-treated rats attenuated all
of the inflammatory responses, such as increased colonic weight-to-body
weight ratio, macroscopic signs of inflammation, increased histological
inflammation score, and MPO activity, which suggest that the
lactoferrin obviously suppressed TNBS-induced colitis. This is the
first study to assess the therapeutic effect of lactoferrin on
TNBS-induced colitis.
In this study, we observed that activation of proinflammatory
cytokines, such as TNF-, IL-1
, and IL-6, in TNBS colitis was suppressed by lactoferrin administration. Many studies have suggested that TNF-
and IL-1
have an important pathogenic role in the development of TNBS-induced colitis through aggregation of
polymorphonuclear cells in the colonic tissue (30). It was
reported that these cytokines were activated in TNBS-induced colitis
and that lethal pancolitis was developed by TNBS administration in
TNF-
transgenic mice, whereas neutralization of TNF-
and IL-1
improved the colitis and chronic TNBS-induced colitis was absent in
TNF-
-knockout mice (27). From these observations, it is
suggested that the anti-inflammatory effect of lactoferrin takes place
by suppression of TNF-
and IL-1
, which were activated in
TNBS-induced colitis.
In contrast to TNF-, IL-1
, and IL-6, IL-4 and IL-10 in the
colonic tissue were activated by lactoferrin. IL-10 was reported to
suppress IL-2 and interferon-
production and to inhibit the synthesis of proinflammatory cytokines such as TNF-
, IL-1
, and IL-6 (9, 33). In addition, it was reported that
IL-10-deficient mice developed chronic enterocolitis (20).
IL-4 also has anti-inflammatory actions. It inhibits the activation of
TNF-
, IL-1
, and IL-6 (37) and suppresses the growth
and migration of lymphocytes and macrophages in the colonic tissue
(36). These observations imply that IL-4 and IL-10 have an
inhibitory effect on colonic inflammation. Our results showed for the
first time that, in inflammatory conditions, oral administration of
lactoferrin significantly increased IL-4 and IL-10 in the colonic
mucosa, whereas in physiological conditions it did not. The activation
of IL-4 and IL-10 by lactoferrin is also considered to contribute to
the anti-inflammatory effect of lactoferrin on TNBS-induced colitis.
Furthermore, NF-B has been demonstrated to play important roles in
colonic inflammation in TNBS-induced colitis and spontaneous colitis of
IL-10-deficient mice (28). It has been proven that cytokines are extremely important regulators of cellular infiltration, tissue damage, ulceration, secretion/diarrhea, motility, and fibrosis (19). The results of our study provide evidence that
complex interactions among cytokines in the colonic inflammation exceed superficial response and that the immunomodulatory effect of
lactoferrin on colonic inflammation is very attractive for treatment of colitis.
It was recently demonstrated that lactoferrin, administered
intravenously or orally, inhibits the production of TNF- and IL-6 in
a murine model stimulated by lipopolysaccharides in the spleen
(37) and in a rat model of carrageenan-induced
inflammation of the foot pad (42). It was also
demonstrated that locally administered lactoferrin inhibits the
production of TNF-
in a murine model of epidermal inflammation in
the ear (8) and that lactoferrin administered in vitro
inhibits the release of TNF-
and IL-1
from mixed lymphocyte
cultures (7). However, there have been no reports that
indicate that administration of lactoferrin modulates
proinflammatory or anti-inflammatory cytokines in the colon. In
this study, we demonstrate for the first time that administration of
lactoferrin modulates proinflammatory and anti-inflammatory cytokines in the colon, resulting in attenuation and prevention of colitis.
It was recently suggested that NF-B plays an important role in
development of TNBS colitis on the basis of the observation that the
transcription factor from NF-
B was excessively activated in colitis
and that an antisense oligonucleotide to NF-
B abrogated the clinical
and histological signs of colitis associated with the TNBS model
(28). It has been revealed that the signal transduction pathways activated in response to TNF-
and IL-1
initiated NF-
B activation through the activation of I
B kinase, which phosphorylated I
B, the dissociation of I
B and NF-
B, and then the
translocation of NF-
B to the nucleus, allowing NF-
B to play its
role in colitis (12, 28, 35). In this study, our
observations provided evidence that the activated pathway of NF-
B in
TNBS-induced intestinal inflammation in rats was attenuated by
lactoferrin and resulted in a fundamental block to developing colitis.
The results that showed an inhibitory effect of lactoferrin on TNF-
and IL-1
suggest that lactoferrin released from secondary granules
of activated neutrophils at an inflammatory site may provide an
inhibitory feedback mechanism to prevent excessive neutrophil
aggregation and activation (7). Further study is required
to explore the role of internal lactoferrin on modulating colonic inflammation.
Our results showed that the plasma concentration of lactoferrin was significantly increased after oral administration of lactoferrin. This is consistent with the report that, after administration of bovine lactoferrin through stomach tube, the plasma concentration significantly increased in piglets (15). But lactoferrin administered with enema was less effective (data not shown). Therefore, the attenuating effect on colitis by orally administered lactoferrin is mainly due to lactoferrin directly absorbed into plasma.
On the other hand, our results showed that, after oral administration
of lactoferrin, the fecal concentration was also significantly increased, and lactoferrin is reported to have bacteriostatic activity
(38); therefore, it remains possible that the decreased inflammation by orally administered lactoferrin is partly due to a
decrease in the colonic bacterial numbers. However, we showed that oral
administration of lactoferrin at the dose (200 mg · kg1 · day
1) did not
have a significant effect on the total number of colonic bacteria in
this model. This result indicates that bacteriostatic activity of
lactoferrin is not the main cause of anti-inflammatory effects in
colonic inflammation.
In this study, oral administration of lactoferrin, whether in the preventive or treatment mode, attenuates the inflammatory reaction of the colon induced by instillation of TNBS. This observation implies the possibility that lactoferrin, in clinical practice, might contribute not only to maintaining remission but even to inducing remission in patients with inflammatory bowel disease.
In conclusion, the results obtained from the experiments with TNBS-induced colitis in rats, an animal model that has similarities with human inflammatory bowel diseases, support and extend the existence of an important anti-inflammatory action of lactoferrin. Although it is not possible to extrapolate findings from animal models to the clinical situation, these data further suggest that lactoferrin is potentially attractive as a therapeutic strategy for the treatment of inflammatory bowel disease.
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ACKNOWLEDGEMENTS |
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We thank Yoshiko Nagoshi for her excellent technical assistance and helpful discussions.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Togawa, Third Dept. of Internal Medicine, Yokohama City Univ. School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan (E-mail: j_togawa{at}med.yokohama-cu.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.
First published February 27, 2002;10.1152/ajpgi.00331.2001
Received 26 July 2001; accepted in final form 20 February 2002.
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