1Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka 565-0871; and 2Department of Gynecology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan
Submitted 29 October 2003 ; accepted in final form 26 June 2004
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ABSTRACT |
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interleukin-8
Interleukin (IL)-8, one of the proinflammatory cytokines, has been demonstrated in many previous studies to be expressed by many different organs in response to inflammatory stimulation (3, 19, 31). It has recently been reported that the IL-1-mediated activation of the IL-8 gene can be regulated in a cell type-specific manner, and that in intestinal cell lines, the transcriptional nuclear factor
B (NF-
B)-responsive element regulates the inducible activity of the IL-8 promoter in response to IL-1
stimulation (39). In most cell lines, NF-
B exists in an inactive state constitutively in the cytoplasm, bound to inhibitory proteins (members of the I
B family). When activated, NF-
B dissociates from its inhibitor, translocates to the nucleus, binds to NF-
B binding sites in target genes such as proinflammatory cytokines, and stimulates their transcription (20). Various bacterial cell wall components, including lipopolysaccharide (LPS), also act as activators of the NF-
B pathway (35), and this signal transduction pathway is known to result in induction of the transcription of IL-8 (38). Several pathways leading to NF-
B activation have been investigated, and the major pathway involves the activation of the I
B kinase (IKK) complex, which leads to the phosphorylation of I
B and to its degradation by the ubiquitin-proteasome system (34). Both IL-1
and LPS use the same signaling pathway downstream of IKK. Claud et al. (9) reported recently that in several types of intestinal epithelial cells, the stimulation of IL-8 secretion by IL-1
was decreased by the addition of several factors found in human breast milk (16, 18), such as transforming growth factor-
(TGF-
), erythropoietin (Epo), IL-10, and epidermal growth factor (EGF).
We show in this report that human breast milk dramatically suppresses inducible IL-8 promoter gene activation in human intestinal cells by inhibiting the activation of NF-B. Moreover, human breast milk can reduce the activation of NF-
B via an I
B
-associated pathway. We also show significant induction of I
B
in the cytoplasm by stimulation with human breast milk. These results suggest a possible mechanism by which human breast milk protects neonates from inflammatory bowel diseases such as NEC by suppressing the NF-
B signaling pathway.
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MATERIALS AND METHODS |
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Preparation of breast milk. Human breast milk was provided by six puerperal patients, each of whom had delivered a full-term infant without medical complications, after they provided informed consent. Milk samples were donated 24 days after parturition. As described previously (37), the samples were stored at 4°C, handled within 24 h of expression, and then pooled. We centrifuged the milk samples twice (12,000 rpm for 10 min at 4°C) to remove the fat component and cellular debris, and used only the whey for the experiments. The aqueous layer was stored at 20°C until it was assayed. To inactivate heat-sensitive proteins and peptides in human breast milk, we boiled the aqueous layer of human breast milk for 5 min, centrifuged it once (12,000 rpm for 10 min at 4°C), and obtained the supernatant.
IL-8 ELISA.
Cells were seeded onto six-well plates at a density of 1 x 105 cells/well. The culture medium was then changed, and the cells were pretreated with a 5% volume of human breast milk obtained as described above or without any additive. Subsequently, 5 ng/ml of IL-1 (Sigma-Aldrich, St. Louis, MO) was added to the culture medium, and 6 h after addition of this cytokine, the supernatants of the culture medium were collected and the concentration of secreted IL-8 was determined using ELISA. Cells were also pretreated with or without a 5% volume of breast milk and then treated with 10 µg/µl of LPS (Sigma-Aldrich) for 24 h as described previously (22), and the IL-8 concentration in the culture medium was measured by performing ELISA. In other experiments, cells were pretreated for 24 h with either a 5% volume of breast milk or boiled breast milk obtained as described above, and IL-8 secretion into the culture medium of cells that were then supplemented with 5 ng/ml IL-1
for 6 h was measured using ELISA. Furthermore, cells were pretreated with breast milk in the presence of 1 ng/µl of anti-IL-10 antibody (Techne, Minneapolis, MN), 0.5 µg/µl of anti-human EGF antibody (R&D Systems, Minneapolis, MN), or 5 ng/µl of anti-Epo antibody (R&D Systems) and subsequently stimulated with 5 ng/ml of IL-1
. The concentration of IL-8 secreted into the culture medium was determined using ELISA. A human IL-8 ELISA kit (R&D Systems) was used to quantify cytokine levels as recommended by the manufacturer. All experiments were repeated three times, and each sample was assayed in duplicate. The data are presented as the concentration of IL-8 in picograms per 105 cells (means ± SD).
Plasmids and plasmid construction.
The IL-8 promoter luciferase plasmid [(wt)LUC] and IL-8 promoter luciferase plasmids with site-directed mutations of activator protein-1 (AP-1), CCAAT/enhancer binding protein (C/EBP), and NF-B binding elements [(mAP-1)LUC, (mC/EBP)LUC, and (mNF-
B)LUC, respectively] were provided to us by Dr. X. Wen (Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA) (39). The NF-
B-dependent promoter construct and its mutant plasmid, which contains four repetitions of the NF-
B binding element (4x
Bw-LUC) and a mutated plasmid thereof (4x
Bm-LUC), were provided to us by Dr. T. Okamoto (Dept. of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan) (24). The structures of these luciferase constructs are illustrated in Fig. 1.
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SDS-PAGE and Western blot analysis.
Caco-2 cells were cultured in 10-cm dishes with complete medium (20% FBS) until 90% confluence, either a 5% volume of human breast milk or nothing was added to the culture medium, and then the cell cultures were further incubated for 24 h. During this additional incubation, the cells were stimulated with IL-1 (5 ng/ml) for various periods as indicated. After stimulation, the cells were harvested and lysed by incubation for 60 min in 100 µl of lysis buffer as described previously (36). These protein samples were separated by performing SDS-PAGE (25 µg/lane) and analyzed by blotting with anti-IL-1 soluble receptor type 1 antibody (Sigma-Aldrich), anti-phospho-IKK
/IKK
antibody, anti-IKK
antibody, anti-IKK
antibody, anti-I
B
antibody, anti-phospho-I
B
antibody (Cell Signaling Technology, Beverly, MA), or anti-
-actin antibody (Sigma-Aldrich). Subsequently, the proteins were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ), and rehybridization was performed using a Reblot Western blot recycling kit (Chemicon International, Temecula, CA).
Statistical analysis. Statistical analysis was performed using the Kruskal-Wallis test, and P < 0.05 was accepted as statistically significant.
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RESULTS |
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Human breast milk can increase the expression of IB
protein.
Next, we assumed that increasing the basal amount of I
B
protein might lead to more extensive interaction with NF-
B proteins and thereby repress their activation. Thus we examined whether human breast milk affected the expression of the basal level of I
B
protein. The total amount of I
B
protein was significantly increased after treatment with human breast milk compared with that in the untreated condition (Fig. 9). This suggests that increasing the basal amount of I
B
protein is also one of the possible mechanisms by which human breast milk exerts its suppressive effect on the NF-
B signaling pathway.
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DISCUSSION |
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IL-8 has been a good model of the immunological response to inflammation in many previous studies, especially in intestinal epithelial cells (25). Also, IL-1 is considered to be useful in these studies as an endogenous inflammatory stimulant that upregulates IL-8 secretion as an innate response of enterocytes (15, 33). In other studies, in vivo evidence that NEC is caused by the coincidence of bacterial infection, intestinal ischemia-reperfusion injury, and systemic inflammation has been obtained using animal models (21). As shown in recent studies by Claud et al. (9), IL-8 secretion from intestinal epithelial cells stimulated by IL-1
was decreased by the addition of several factors found in human breast milk (16, 18), such as TGF-
, Epo, IL-10, and EGF. However, whether human breast milk transcriptionally suppresses the IL-8 gene has never been examined, and the in vitro mechanism of suppression remains unclear. Thus our present study is the first demonstration of the molecular mechanism of the anti-inflammatory effect of human breast milk in vitro on human intestinal epithelial cells. Our findings show that the suppression of the IL-8 promoter by human breast milk occurs via the inhibition of the activation pathway of NF-
B.
TNF and LPS are also well known as physiological inflammatory stimulators and potent NF-
B activators. Consistent with findings in two previous studies (22, 32), Caco-2 cells responded to IL-1
and LPS in our experiments. We have shown that human breast milk suppressed both IL-1
- and LPS-induced IL-8 secretion (Fig. 2) as well as IL-8 promoter activation (Fig. 4 and unpublished data). However, as also previously reported by Eckmann et al. (13), we have shown in luciferase assays using IL-8 promoter that Caco-2 cells did not respond to TNF
(our unpublished data).
As reviewed in a previous report (7), platelet-activating factor (PAF) is another factor that is known to play an important role in neonatal intestinal injury. PAF, TNF, and LPS act synergistically to amplify inflammation (21), and PAF enhances the DNA binding activity of NF-
B in the intestine, predominantly as p50 subunits (12). Human breast milk may also exert an inhibitory effect on PAF-induced intestinal inflammation by suppressing the activation pathway involving NF-
B. Further examination is needed to test this possibility.
Our findings suggest that there may be two different mechanisms by which human breast milk exerts its inhibitory effect on NF-B activity, namely, through the regulation of both the production and phosphorylation of I
B
proteins. One possibility is that an increased basal quantity of I
B
protein caused by human breast milk may lead to increased interaction between I
B
and NF-
B, which may in turn prevent the activation of NF-
B protein. We can also suggest another possibility, namely, that human breast milk exerts its inhibitory effect by directly suppressing the phosphorylation of I
B
protein. As shown in our study, human breast milk did not decrease the protein level of IL-1R (Fig. 6), which occurs in the first step of the IL-1
-induced NF-
B signaling pathway. Furthermore, we also have shown that the IL-1
-induced phosphorylation of IKK
/IKK
was not affected by human breast milk (Fig. 7). Therefore, the target of the inhibitory effect of human breast milk on NF-
B signaling is downstream of IKK.
It is well known that phosphorylation of IB
leads to its ubiquitination and degradation by the 26S proteasome, thus leading to NF-
B nuclear translocation (34). Hypoxia-inducible factor-1
(HIF-1
) is also a well-known transcriptional factor that is regulated by the ubiquitin-proteasome degradation pathway (26). HIF-1
is activated under conditions of reduced oxygen and regulates the transcription of several genes that are responsive to a lack of oxygen, such as Epo, vascular endothelial growth factor, and glucose transporter 1. In a previous report (14), the parallel induction of HIF-1
and intestinal trefoil factor (ITF) was observed under hypoxic conditions in the Caco-2 cell line. ITF is a protein that has a protective role against reduced blood flow in the intestine and works to preserve the barrier function of the intestine against outer stimuli, and thus HIF-1
may have a protective role against the ischemic changes that occur in the neonatal intestine in disease conditions such as NEC. In our previous studies, we proved that human breast milk caused significant induction of HIF-1
proteins in the nuclei of Caco-2 cells (our unpublished data). The inhibitory effect of human breast milk may be associated with suppression of the ubiquitination and degradation pathway and may cause an increased level of proteins with a protective role against intestinal ischemia-reperfusion injury and systemic inflammation, such as HIF-1
or I
B
.
Glucocorticoids are among the most potent agents whose effects of anti-inflammation and immunosuppression are widely accepted, and they are commonly used for the treatment of inflammatory bowel diseases such as Crohns disease or ulcerative colitis. They are considered to inhibit the synthesis of cytokines necessary for the immune response, and as proved in several previous studies (2, 6), glucocorticoids exert their inhibitory effect against the activation of NF-B by suppressing the induction of I
B
. Human breast milk contains glucocorticoids, and they are among the candidate breast milk components that showed an inhibitory effect in our experiments. As shown by our findings, the repressive effect of human breast milk was diminished when the milk was boiled (Fig. 3). Thus some unknown substance in human breast milk that is sensitive to heat, like most proteins, exerts this inhibitory effect. We also have shown that the suppressive effect of human breast milk on IL-1
-induced IL-8 secretion was reduced in the presence of neutralizing antibodies to IL-10, EGF, or Epo (Fig. 3). However, the suppressive effect of breast milk was not totally abrogated; instead, the effect of boiling on these neutralizing antibodies was partial. We are now attempting to identify this unknown factor with the hope that it may be therapeutic for NEC in the future.
As reported in previous studies (4, 11), longer breastfeeding may also have a protective effect against childhood acute leukemia and lymphoma. The detailed mechanism of this phenomenon has not been fully clarified, but it was suggested in another report that NF-B may play a determining role in the sensitivity or resistance to the progression of anaplastic large cell lymphoma or Hodgkin disease (HD) (30). In that study, HD cells were sensitized by ectopic overexpression of I
B. Therefore, our present findings can explain the mechanism by which human breast milk exerts its protective effect against childhood acute leukemia or lymphoma, and the unknown proteins in human breast milk that suppress NF-
B signaling may also be therapeutic agents for these diseases. In conclusion, further studies are needed before applying the inhibitory factors in human breast milk as therapeutic agents in humans for the prevention of inflammatory bowel diseases such as NEC or for the treatment of childhood lymphoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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