Hiroshi Kono, Hideki Fujii, Masami Asakawa, Akira Maki, Hidetake Amemiya, Yu Hirai, Masanori Matsuda and Masayuki Yamamoto Am J Physiol Gastrointest Liver Physiol 286:1081-1089, 2004. doi:10.1152/ajpgi.00457.2003 You might find this additional information useful... This article cites 37 articles, 16 of which you can access free at: http://ajpgi.physiology.org/cgi/content/full/286/6/G1081#BIBL Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl on the following topics: Agriculture .. Corn Biochemistry .. Triglycerides Biochemistry .. Corn Oil Physiology .. IgA Oncology .. Immune Response Physiology .. Rats Updated information and services including high-resolution figures, can be found at: http://ajpgi.physiology.org/cgi/content/full/286/6/G1081

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AJP - Gastrointestinal and Liver Physiology publishes original articles pertaining to all aspects of research involving normal or abnormal function of the gastrointestinal tract, hepatobiliary system, and pancreas. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. ISSN: 0193-1857, ESSN: 1522-1547. Visit our website at http://www.the-aps.org/.

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Am J Physiol Gastrointest Liver Physiol 286: G1081–G1089, 2004; 10.1152/ajpgi.00457.2003.

Medium-chain triglycerides enhance secretory IgA expression in rat intestine after administration of endotoxin Hiroshi Kono,1 Hideki Fujii,1 Masami Asakawa,1 Akira Maki,1 Hidetake Amemiya,1 Yu Hirai,1 Masanori Matsuda,1 and Masayuki Yamamoto2 1

First Department of Surgery, University of Yamanashi, Yamanashi 409-3898; and 2Department of Surgery, Shinko Byoin Hospital, Hyogo 651-0072, Japan Submitted 24 October 2003; accepted in final form 20 January 2004

saturated fat; inflammatory cytokines; endotoxemia; chemokines; Peyer’s patch

the “motor” responsible for multiple organ failure in critical illnesses (18). The gut mucosa is the site in which various acute phase proteins (23, 36, 37), gut hormones (38), and cytokines (22) are produced that affect the mucosa as well as the function and integrity of remote organs and tissues. Thus the role of the intestinal mucosa in inflammatory and metabolic responses to sepsis and endotoxemia has become more apparent during the past decade. We reported that a daily supplement of medium-chain triglycerides prevented an increase in gut permeability and intestinal injury after endotoxin administration (11, 12). We speculated that medium-chain triglycerides help protect the gut during endotoxemia or sepsis. Proinflammatory cytokines such as TNF-␣ are involved in triggering a vicious cycle in infec-

tious insults via the redox-sensitive transcriptional factor, NF-␬B (14). These cytokines upregulate chemoattracting factors, which subsequently increase the number of leukocyte into the host tissue. Thus inflammatory cytokines and chemokines are involved in organ injury. Alternatively, secretory IgA is synthesized within the lamina propria through the interaction of T and B cells and then transported by overlying epithelial cells onto the mucosal surface using secretory components. There, it binds to specific antigens on bacteria (16, 31), viruses, and other toxic molecules and prevents them from attaching to the mucosal surface. It also plays a role in eliminating infectious agents that have penetrated epithelial cell layers. Intestinal secretory IgA levels correlate inversely with bacterial overgrowth, bacterial translocation, and changes in intestinal permeability in animal models (4). Thus secretory IgA plays an important role in protecting against infection in the intestinal immune system (20, 21). The purpose of this study was to determine whether medium-chain triglycerides enhance the production of secretory IgA and inhibit expression of inflammatory mediators in the intestine. The production of secretory IgA is controlled by cytokine-producing T cells within the gut-associated lymphoid tissue and possibly by cytokines released from the mucosa. T cells, which are major producers of cytokines, are classified into two distinct subsets, Th1 and Th2, based on the pattern of cytokines they secrete. Th1 cytokines such as IFN-␥ and TNF-␤ downregulate IgA production, whereas Th2 cytokines such as IL-4, IL-5, IL-6, and IL-10 upregulate IgA production (15). A balance between Th1 and Th2 cytokines may be necessary to maintain a normal IgA immune response. Therefore, mRNA expression of IFN-␥ and IL-10 in the ileum and Peyer’s patches was also investigated.

THE GUT MUCOSA HAS BEEN CALLED

Address for reprint requests and other correspondence: H. Kono, First Dept. of Surgery, Univ. of Yamanashi, 1110 Shimokato, Tamaho, Nakakoma, Yamanashi 409-3898, Japan (E-mail: [email protected]). http://www.ajpgi.org

MATERIALS AND METHODS

Animals and treatments. Male Sprague-Dawley rats (250 –300 g body wt; Japan SLC, Shizuoka, Japan) were used in this study. They were housed under barrier-sustained conditions and allowed to recover for at least 5 days after arrival before being used. During this acclimation period, rats were fed Purina rat chow (cat. no. 5001; Dyets, Bethlehem, PA). The experimental protocol followed the institutional and the National Research Council criteria for the care and use of laboratory animals in research. Furthermore, all rats received humane care in compliance with institutional guidelines. Rats were given medium-chain triglycerides (saturated fat, trioctanoin 8:0; 5 g/kg, Nihon-Yushi, Tokyo, Japan) or the same dose of corn oil (polyunsaturated fat) by gavage daily for 1 wk (11), and received food and water ad libitum throughout the study. Previous 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.

0193-1857/04 $5.00 Copyright © 2004 the American Physiological Society

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Kono, Hiroshi, Hideki Fujii, Masami Asakawa, Akira Maki, Hidetake Amemiya, Yu Hirai, Masanori Matsuda, and Masayuki Yamamoto. Medium-chain triglycerides enhance secretory IgA expression in rat intestine after administration of endotoxin. Am J Physiol Gastrointest Liver Physiol 286: G1081–G1089, 2004; 10.1152/ajpgi.00457.2003.—The purpose of this study was to determine whether medium-chain triglycerides (MCTs) modulate the inflammatory immune response to LPS and enhance the expression of secretory IgA in the rat intestine. Rats were given either corn oil or MCTs by gavage daily for 1 wk, and LPS or saline vehicle was administered via the tail vein. They were then killed, and serum and sections from the gut were collected for further analysis. Western blot analysis for secretory IgA revealed that MCTs significantly enhanced its expression in the ileum compared with corn oil in rats administered saline. After LPS challenge, expression of secretory IgA was decreased in the corn oil group but not in the MCTs group. The mRNA expression of IL-6 was assessed by real-time RT-PCR, because IL-6 regulates secretory IgA in the intestine. The expression was significantly greater in the MCTs group than in the corn oil group after LPS injection. Increases in expression of proinflammatory cytokines or chemokines such as TNF-␣, IL-18, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 in the ileum were significantly blunted by MCTs. In addition, the mRNA expression of the Th2 IgA-stimulating cytokine IL-10 in the ileum and Peyer’s patches was significantly greater in the MCTs than the corn oil group. In contrast, the mRNA expression of the Th1 IgA-inhibiting cytokine interferon-␥ was blunted by MCTs. As a result, intestinal injury was significantly reduced. Therefore, MCTs protect the gut by modulating the immune response to LPS and enhancing secretory IgA expression.

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secretory IgA. Samples of bile and intestinal crude particulate fractions were diluted in nonreducing Laemmli buffer. Extracted proteins (20 ␮g) were separated by 100 g/l SDS-PAGE and transferred to polyvinylidine fluoride membranes. The membranes were blocked with Tris-buffered saline-Tween 20 containing 50 g/l skim milk and probed with polyclonal goat anti-rat secretory IgA antibodies (ICN) or ␤-actin (Sigma) followed by horseradish peroxidase-conjugated secondary antibody when appropriate. They were then incubated with an enhanced chemiluminescent substrate (ECL reagent; Amersham Life Science, Arlington Heights, IL) and exposed to X-OMAT film (Eastman Kodak, Rochester, NY). Densitometric analysis of the image was performed on a Macintosh computer using NIH image 1.54 analysis software. Real-time RT-PCR for mRNA expression of IL-6, TNF-␣, and IL-18 in mucosa from the ileum; IFN-␥, and IL-10 in the ileum; and Peyer’s patches. mRNA was quantified by real-time RT-PCR (TaqMan; PE Applied Biosystems, Foster City, CA). Real-time RT-PCR was performed by using a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). Predeveloped TaqMan assay reagents (PE Applied Biosystems) were used for sequencing specific primers for IFN-␥ (GenBank accession no. AF010466), IL-10 (GenBank accession no. X60675; L02926), IL-6 (GenBank accession no. M26744), TNF-␣ (GenBank accession no. NM 012676) and IL-18 (GenBank accession no. AJ222813). 18s rRNA was used as an internal control. Four hours after LPS or saline injection, sections of the ileum and Peyer’s patches near the terminal ileum were collected by using scissors for measurement of the mRNA expression of IFN-␥ or IL-10. Nine hours after LPS or saline administration, sections of mucosa from the ileum were collected to measure the mRNA expression of IL-6, TNF-␣, and IL-18. Samples were stored at ⫺80°C until assayed. Total RNA was isolated from 25-mg pieces of liver tissue using a RNA purification kit (Qiagen, Hilden, Germany). Reverse transcription of total RNA (2 ␮g) was performed in a final volume of 100 ␮l containing 1⫻ TaqMan RT buffer, 5.5 mM MgCl2, 500 ␮M of each dNTP, 2.5 ␮M random hexamers, 0.4 ⫻ 106 U/l RNase inhibitor, and 1.25 ⫻ 106 U/l multiscribe RT. cDNA samples (2 ␮l) were used for quantitative RT-PCR (a 10-min step at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C in the presence of specific forward and reverse primers on a TaqMan Universal PCR Master Mix; PE Applied Biosystems). mRNA levels were calculated by using the comparative parameter threshold cycle (Ct) method (6) and normalized to rRNA. To confirm the amplification specificity, the PCR products were subjected to a melting-curve analysis. Semiquantative RT-PCR for the mRNA expression of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 in the ileum. Macrophage inflammatory protein (MIP-2) and monocyte chemoattractant protein (MCP-1) are chemoattractant factors predominantly produced by macrophages. They increase the number of macrophages/monocytes and neutrophils recruited to the inflammatory focus and cause tissue injury. Because predeveloped primer and probe for MIP-2 and MCP-1 were not commercially available, the mRNA expression was assessed by semiquantitative RT-PCR. Intestinal tissue samples were collected from animals killed 4 or 9 h after LPS or saline injections. Total RNA was isolated from 25-mg pieces of liver tissue using a RNA purification kit (Qiagen) according to the manufacturer’s instructions and used for PCR assay to detect the mRNA expressions of MIP-2 and MCP-1. Reverse transcription of total RNA was performed by using the method described above. PCR primers for MIP-2 (10), MCP-1 (33), and GAPDH contained the following sequences: MIP-2 sense (5⬘CAGAGCTTGAGTGTGACG-3⬘) and antisense (5⬘-TCGTACCTGATGTGCCTC-3⬘); MCP-1 sense (5⬘-TCCACCACTATGCAGGTCTC-3⬘) and antisense (5⬘-TGGACCCATTCCTTATTGGG-3⬘); and GAPDH sense (5⬘-TGAAGGTCGGAGTCAACGGATTTGGT3⬘) and antisense (5⬘-CATGTGGGCCATGAGGTCCACCAC-3⬘). Aliquots (5 ␮l) of synthesized cDNA were added to 45 ␮l of PCR mix containing 5 ␮l of 10⫻ PCR buffer, 1 ␮l of each deoxynucleotide

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studies (30) with rats of the same strain, age, and weight showed that their ad libitum intake of chow was 307 kcal 䡠 kg⫺1 䡠 d⫺1. The calorie intake from the daily fat supplement was 45 kcal 䡠 kg⫺1 䡠 d⫺1. During this experiment, a steady increase in weight was observed, and there were no significant differences compared with untreated rats that were also fed chow. No complications were observed in rats given mediumchain triglycerides. After 1 wk, rats were administered either a saline vehicle or LPS (10 mg/kg, Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO) via the tail vein, and survival was assessed over the following 24 h (n ⫽ 8 in each group). Blood sampling and measurement of serum secretory IgA levels. In separate experiments, rats were given medium-chain triglycerides (5 g 䡠 kg⫺1 䡠 d⫺1) or the same dose of corn oil for 1 wk (12). Then, either LPS (10 mg/kg) or a saline vehicle was administered via the tail vein. Blood samples were collected from the aorta 9 h later and centrifuged at 1,200 g for 10 min at 4°C. Serum was stored at ⫺80°C until assayed for secretory IgA. Serum secretory IgA was measured by an ELISA using polyclonal goat anti-rat secretory IgA antibody (ICN, Costa Mesa, CA), and data were corrected for each dilution. Pathology and histological measurement. Sections of gut, liver, and lung were collected 9 h after LPS administration and stained with hematoxylin-eosin to assess inflammation and necrosis. Pathology was evaluated in a blind manner by one of the authors and by an expert in rodent pathology. Furthermore, an observer unaware of the treatment status of the animals from which the tissues were harvested measured 15 villi per slide under a microscope using a micrometer. Immunohistochemistry of secretory IgA in gut. Paraffin-embedded sections of jejunum, ileum, and colon were deparaffinized, rehydrated, and stained immunohistochemically with polyclonal goat anti-rat secretory IgA antibody (ICN) by sequential incubation with polyclonal antibodies (StressGen Biotechnologies, Victoria, BC, Canada) in PBS (pH 7.4) containing 10 g/l Tween 20 and 10 g/l BSA (13). Peroxidase-linked secondary antibody and diaminobenzidine (peroxidase envision kit; DAKO, Carpinteria, CA) were used to detect specific binding. The slides were rinsed twice with PBS-1 g/l Tween 20 between each incubation, and sections were counterstained with hematoxylin as described elsewhere (5). Sections from the same rats were processed without the primary antibody and then examined using the procedure detailed above as a control for nonspecific binding of the secondary antibody. Preparation of samples and immunoblot analysis of IgA. To collect bile as a positive control (2, 8, 28), the bile duct was cannulated with a small length of polyethylene-10 tubing, and bile samples were collected and immediately placed on ice. Cold nonreducing Laemmli buffer (62.5 mM Tris 䡠 HCL, pH 6.8, containing 20 g/l SDS and 0.1 l/l glycerol) containing 12.5 mM benzamidine and 21 mM leupeptin (Sigma) was added to the bile (9:1 vol buffer/bile). The resulting mixture was stored at ⫺80°C. Tissue samples of the ileum, jejunum, and colon were collected from rats 9 h after saline or LPS administration. Intestinal tissue was washed free of its contents using cold PBS, blotted on filter paper, and weighed. A crude particulate fraction of the intestine was prepared according to the method of Perez et al. (26) with some modifications. Briefly, the organ was homogenized by using a homogenizer (Caframo, Wiarton, ON, Canada). Ten volumes of cold buffer (pH 7.4) were used containing in mM: 10 TES (Sigma), 1 MgCl2, 12.5 benzamidine, and 21 leupeptin plus 0.2 M sucrose. A small sample of homogenate was kept to assay the protein concentration. The remainder was centrifuged at 1,000 g for 10 min at 4°C after which the supernatant was ultracentrifuged at 100,000 g for 60 min at 4°C. The resulting pellet was resuspended in TES-sucrose buffer containing 10 ml/l Triton X-100, 12.5 mM benzamidine, 21 mM leupeptin, and 1 mM PMSF (Boehringer-Mannheim, Mannheim, Germany). This procedure yielded a crude particulate fraction rather than a pure membrane fraction. It was stored at ⫺80°C. Western blot analysis of secretory IgA in the gut. Intestinal tissues were collected 9 h after LPS or saline administration for expression of

MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY

Table 1. Effect of LPS and MCT on mortality Saline, 1 ml/kg

Mortality

LPS, 10 mg/kg

CO (5 mg/kg)

MCT (5 mg/kg)

CO (5 mg/kg)

MCT (5 mg/kg)

0

0

100*

0†

Values are % mortality of 8 rats given 5 mg/kg of corn oil (CO) or medium-chain triglycerides (MCT). Mortality after LPS administration was determined as described in MATERIALS AND METHODS. *P ⬍ 0.05 compared with rats given a saline vehicle; †P ⬍ 0.01 compared with rats given CO with LPS by Fisher’s exact test.

RESULTS

Effect of lipid type and LPS on mortality. All animals survived 24 h after saline administration in both groups (Table 1). However, all animals given corn oil appeared moribund 9 h after LPS injection and died within 24 h after LPS adminis-

tration (Table 1). Mortality was absent in rats given mediumchain triglycerides. Effect of lipid type and LPS on the gut. Complications were not observed in rats given either medium-chain triglycerides or corn oil for 1 wk. Furthermore, there were no significant differences in body weight between the two groups. Morphological changes were not observed in the gut sections from rats treated with corn oil for 1 wk after saline injection (Fig. 1). In contrast, medium-chain triglycerides significantly increased the length of villi (normal rats, 755 ⫾ 92 ␮m; rats given corn oil, 762 ⫾ 82 ␮m; and rats given medium-chain triglycerides, 802 ⫾ 88 ␮m, P ⱕ 0.05). Furthermore, the number of goblet cells in the gut also increased, which was consistent with the results of a previous study (12). After LPS administration, the ileum from rats given corn oil showed hemorrhagic changes in the submucosal layer and necrosis of the epithelial cells. However, these changes were almost completely prevented by medium-chain triglycerides. Furthermore, liver (12) and lung injury was also prevented by medium-chain triglycerides (data not shown). Effect of lipid type and LPS on serum secretory IgA levels. Serum secretory IgA was not significantly different between the two groups 9 h after saline administration (Fig. 2). After LPS injection, values were decreased by ⬃40% in rats given corn oil. However, levels increased ⬃1.5-fold in rats given medium-chain triglycerides. Effect of lipid type and LPS on intestinal secretory IgA levels. Secretory IgA expression was assessed in the gut by immunohistochemistry (Fig. 3). In rats administered saline, the expression was minimal in each intestinal section. Although LPS administration increased the expression in each gut sec-

Fig. 1. Effect of lipid type and LPS on the gut. Sections of the distal ileum and colon from rats given a saline vehicle (VEH) [A, corn oil (CO); B, medium-chain triglycerides (MCT)] and LPS; (C, CO; D, MCT) are shown. Black arrows indicate submucosal hemorrhage and white arrows indicate necrosis. Original magnification, ⫻400. Representative photomicrographs from 6 rats/group.

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(1 mM each), 0.5 ␮l of sense and antisense primers (0.15 mM), and 0.25 ␮l of DNA polymerase (Gene Amp PCR kit; Perkin Elmer Cetus, Norwalk, CT). The amplified PCR products were electrophoresed at 100 volts through a 2% agarose gel (BRM, Sigma) for ⬃30 min. The gels were stained with 0.5 mg/ml ethidium bromide Tris-borate-EDTA buffer (ICN) and photographed with Type 55 Polaroid positive/negative film. Densitometric analysis of the captured image was performed on a Macintosh computer using NIH image 1.54 analysis software. The area under the curve was normalized for GAPDH content. Statistics. Data are expressed as the means ⫾ SE. ANOVA with Bonferroni’s post hoc test or the Student’s t-test was used to determine significance when appropriate. A P value ⬍ 0.05 was considered significant.

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Fig. 3. Immunohistochemical detection of sIgA in the gut. Nine hours after LPS administration, sections from jejunum, ileum, and colon from rats given corn oil or medium-chain triglycerides were stained by using a mouse anti-rat sIgA antibody (brown staining) as detailed in MATERIALS AND METHODS. Original magnification, ⫻200. Representative photomicrographs. AJP-Gastrointest Liver Physiol • VOL

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Fig. 2. Effect of lipid type and LPS on serum secretory IgA (sIgA). Serum samples were collected 9 h after LPS or saline vehicle administration and sIgA was measured by ELISA as detailed in MATERIALS AND METHODS. Values are the means ⫾ SE (n ⫽ 8). *P ⬍ 0.05 compared with rats given corn oil with a saline vehicle; and #P ⬍ 0.05 compared with rats given medium-chain triglycerides with vehicle by ANOVA with Bonferroni’s post hoc test.

tion in both treatment groups, it was greatest in the ileum. Furthermore, elevated expression was observed in all intestinal sections from rats given medium-chain triglycerides (Fig. 3). To confirm these results, Western blot analyses of secretory IgA and ␤-actin were performed on intestinal samples (Fig. 4, A and B). The expression was greatest in the ileum, followed by the colon, in untreated normal rats (Fig. 4B). Further analysis was carried out by using samples from the ileum. After saline injection, the expression did not change in rats given corn oil. However, in rats given medium-chain triglycerides, it was significantly increased ⬃1.2-fold compared with the corn oil group. Furthermore, after LPS administration, the expression decreased significantly by ⬃60% in rats given corn oil but not in those given medium-chain triglycerides. mRNA expression of IL-6, TNF-␣, and IL-18 in the mucosa of the ileum. The mRNA expression of IL-6, TNF-␣, and IL-18 was assessed in the ileum from rats treated with corn oil or medium-chain triglycerides (Fig. 5). Nine hours after saline administration, the mRNA expression of IL-6 was greater in rats given medium-chain triglycerides than in those given corn oil. Although LPS administration significantly increased the mRNA expression of IL-6 in both groups, it was significantly greater in rats given medium-chain triglycerides compared with those given corn oil. After saline administration, the mRNA expression of TNF-␣ and IL-18 was not significantly different between the two groups. However, the expression of both cytokines increased approximately twofold in rats given corn oil after LPS administration. The expression was significantly blunted in the medium-chain triglyceride group.

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Effect of lipid type and LPS on MIP-2 and MCP-1 mRNA expression in the ileum. After saline administration, the mRNA expression of MIP-2 was significantly greater in the ileum of rats given corn oil compared with those given medium-chain triglycerides (Fig. 6). After LPS administration, the expression significantly increased in rats given corn oil, whereas the increase was significantly blunted in rats treated with mediumchain triglycerides. After saline administration, the mRNA expression of MCP-1 was not detected in either group. In contrast, LPS increased significantly the expression of MCP-1 in rats given corn oil, and the increase was significantly blunted in the medium-chain triglyceride group. The same results were observed in samples from rats administered LPS after 9 h (data not shown). mRNA expression of IFN-␥ and IL-10 in the ileum and Peyer’s patches. The mRNA expression of Th1 IgA-inhibiting cytokine IFN-␥ and Th2 IgA-stimulating cytokine IL-10 was assessed in the ileum and Peyer’s patches from rats treated with corn oil or medium-chain triglycerides (Fig. 7). Nine hours after saline administration, the mRNA expression of IFN-␥ was significantly less in rats given medium-chain triglycerides compared with those given corn oil. In contrast, LPS administration significantly increased expression of IFN-␥ in rats given corn oil, and this increase was blunted by ⬃30% by medium-chain triglycerides. Medium-chain triglycerides significantly increased the mRNA expression of IL-10 in the ileum but not in the Peyer’s patches of rats administered saline. After LPS administration, the expression did not change significantly in rats given corn oil. However, it increased significantly ⬃1.5- to 2-fold in rats given medium-chain triglycerides. DISCUSSION

Effects of medium-chain triglycerides on the intestinal inflammatory immune response in endotoxemia. Macrophages are an important part of the immunological and inflammatory

Fig. 5. The mRNA expressions of IL-6 (A), TNF-␣ (B), and IL-18 (C) in the ileum. Tissue samples were collected 4 h after LPS or saline administration and the mRNA expression of cytokines was measured by real-time RT-PCR as detailed in MATERIALS AND METHODS. mRNA levels were calculated by using the comparative parameter threshold cycle (Ct) method and normalized to ribosomal RNA. Data represent the means ⫾ SE (n ⫽ 6). *P ⬍ 0.01 compared with rats given corn oil with a saline vehicle; #P ⬍ 0.05 compared with rats given corn oil with LPS by ANOVA with Bonferroni’s post hoc test. AJP-Gastrointest Liver Physiol • VOL

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Fig. 4. Effect of LPS and lipid type on the expression of sIgA in the gut. Protein extracts (50 ␮g) from the gut were analyzed by Western blotting using a mouse anti-rat sIgA antibody. A: specific bands for sIgA. B: densitometric analysis (n ⫽ 4 in each group). *P ⬍ 0.01 compared with rats given corn oil with a saline vehicle; #P ⬍ 0.05 compared with rats given corn oil with LPS by ANOVA with Bonferroni’s post hoc test.

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responses. A large population of these cells resides in the normal intestinal mucosa. In inflammatory bowel disease, the mucosal macrophage population increases and is derived from circulating monocytes. They are phenotypically different from the resident population and play a major role in mediating the mucosal inflammation (19). They predominantly secrete proinflammatory cytokines, such as TNF-␣ and IL-18, which are involved in intestinal injury caused by inflammation (24). IL-18 was a primary mediator of the inflammation associated with dextran sulfate sodium-induced colitis in mice (29). In this study, increases in the mRNA expression of TNF-␣ and IL-18 were blunted by ⬃50% by medium-chain triglycerides (Fig. 5). Among the inflammatory mediators expressed by proinflammatory cytokines are chemoattractant factors, such as MIP-2 and MCP-1. They control the nature and magnitude of inflammatory cell infiltration to the site of inflammation, and they are also involved in organ injury (32). Therefore, the mRNA expression of chemokines was assessed by RT-PCR. Medium-chain triglycerides also inhibited the mRNA expression of both MIP-2 and MCP-1 after LPS administration (Fig. 6). Thus medium-chain triglycerides may inhibit infiltration and activation of inflammatory cells in the gut, leading to decreases in inflammatory cytokine expression during endotoxemia. Among the cytokines produced in the intestinal mucosa during inflammation, IL-6 is important because of its multiple biological effects in the intestine as well as other organs and

tissues. IL-6 is an integral part of the inflammatory response to sepsis and endotoxemia. Under different conditions, IL-6 may exert pro- or anti-inflammatory effects. In conditions of “uncontrolled” inflammation, high IL-6 levels contribute to morbidity and mortality (3). In a previous study, systemic IL-6 levels gradually increased up to 6 h after a lethal dose of LPS, and this level correlated with pathophysiology and mortality (12). Furthermore, levels increased significantly in rats given corn oil and correlated with organ injury and mortality (12). Medium-chain triglycerides, however, prevented these events. In addition to the biological roles of systemic levels of IL-6, it has important biological effects on the intestinal mucosa (27). Mucosal levels of IL-6 regulate enterocyte acute-phase protein synthesis (23), protein synthesis in the mucosa (35), and intestinal secretory IgA production (1). IL-6 is also an important regulator of secretory IgA production by B cells in the Peyer’s patches. In this study, the mRNA expression of IL-6 was enhanced by medium-chain triglycerides before and after LPS administration (Fig. 5). Furthermore, intestinal and serum secretory IgA increased significantly in rats given mediumchain triglycerides compared with those given corn oil after LPS administration (Figs. 2–4). Thus medium-chain triglycerides increase the expression of intestinal IL-6, which possibly correlates with increases in expression of secretory IgA in the gut. The role of intestinal secretory IgA and mucosal immunology during endotoxemia. IgA, but not IgG, antibodies against fecal endotoxins increased significantly in patients with alco-

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Fig. 6. Effect of lipid type and LPS on the mRNA expression of macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1 in the ileum. Tissue samples were collected 4 h after saline or LPS administration, and the mRNA expression of chemokines was measured by semiquantitative RT-PCR as detailed in MATERIALS AND METHODS. A and B: mRNA signals expressed as a ratio to GAPDH mRNA as measured by densitometric analysis. C: amplification of each mRNA transcript. Samples shown are representative of 4 samples/group. Data represent the means ⫾ SE (n ⫽ 6). *P ⬍ 0.01 compared with rats given corn oil with a saline vehicle; #P ⬍ 0.05 compared with rats given corn oil with LPS by ANOVA with Bonferroni’s post hoc test.

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hol-induced liver disease (25). IgA antibodies against fecal endotoxin correlated closely with the plasma concentrations of alanine aminotransferase, ␥-glutamyl transferase, and C-reactive protein in patients with alcoholic liver disease. Because medium-chain triglycerides increased serum and intestinal secretory IgA (Figs. 2– 4) and prevented intestinal (Fig. 1) and liver injury (12) after LPS administration, elevated production of secretory IgA by medium-chain triglycerides contributes to the inactivation of endotoxin. Also, secretory IgA located in body tissue was shown to suppress the inflammatory process, thereby reducing its damaging effects on multiple organs (25). Peyer’s patches are the principal sites responsible for sensitization of secretory IgA. In humans, most of the Peyer’s patches are located in the distal small intestine, which contains microflora similar to the colon due to backwash from the colon through the ileocecal valve (25). The Peyer’s patches were also predominantly located in the distal ileum in rats in this study

(data not shown). mRNA expression of Th2 IgA-stimulating anti-inflammatory cytokines such as IL-10 was significantly induced in this region by glutamine after LPS administration (7). These cytokines stimulate IgA production in vitro by cells of the gut-associated lymphoid tissue. Thus intestinal secretory IgA production is associated with the expression of Th2 IgAstimulating cytokines (17). The mRNA expression of IL-10 was significantly increased in the ileum and Peyer’s patches in rats given medium-chain triglycerides compared with those given corn oil after LPS administration (Fig. 7). Furthermore, the mRNA expression of the Th1 IgA-inhibiting cytokine INF-␥ also decreased in rats given medium-chain triglycerides. Therefore, the Peyer’s patches play an important role as an intestinal lymphoid organ in endotoxemia. Medium-chain triglycerides also affect the expression of secretory IgA, which possibly reduces the inflammatory immune response against endotoxin in the gut.

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Fig. 7. The mRNA expressions of IFN-␥ (A, C) and IL-10 (B, D) in the ileum (C, D) and Peyer’s patches (A, B). Tissue samples were collected 4 h after LPS or saline vehicle administration and the mRNA expression of cytokines was measured by RT-PCR as detailed in MATERIALS AND METHODS. The mRNA levels were calculated by using the comparative Ct method and normalized to ribosomal RNA. Data represent the means ⫾ SE (n ⫽ 6). *P ⬍ 0.01 compared with rats given corn oil with a saline vehicle; #P ⬍ 0.05 compared with rats given corn oil with LPS by ANOVA with Bonferroni’s post hoc test.

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12. Kono H, Fujii H, Asakawa M, Yamamoto M, Matsuda M, Maki A, and Matsumoto Y. Protective effects of medium-chain triglycerides on the liver and the gut in rats administered endotoxin. Ann Surg 237; 246 –255, 2003. 13. Kono H, Nakagami M, Rusyn I, Connor HD, Stefanovic B, Brenner DA, Mason RP, Arteel GE, and Thurman RG. Development of an animal model of chronic alcohol-induced pancreatitis in the rat. Am J Physiol Gastrointest Liver Physiol 280: G1178 –G1186, 2001. 14. Kono H, Rusyn I, Yin M, Gabele E, Yamashina S, Dikalova A, Kadiiska MB, Connor HD, Mason RP, Segal BH, Bradford BU, Holland SM, and Thurman RG. NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease. J Clin Invest 106: 867– 872, 2000. 15. Kramer DR, Sultherland RM, Bao S, and Husband AJ. Cytokinemediated effects in mucosal immunity. Immunol Cell Biol 73: 389 –396, 1995. 16. Kudsk KA. Current aspects of mucosal immunology and its influence by nutrition. Am J Surg 183: 390 –398, 2002. 17. Kudsk KA, Wu Y, Fukatsu K, Zarzaur BL, Johnson CD, Wang R, and Hanna MK. Glutamine-enriched total parenteral nutrition maintains intestinal interleukin-4 and mucosal immunoglobulin A levels. JPEN J Parenter Enteral Nutr 24: 270 –274, 2000. 18. Langkamp-Henken B, Donovan TB, Pate LM, Maull CD, and Kudsk KA. Increased intestinal permeability following blunt and penetrating trauma. Crit Care Med 23: 660 – 664, 1995. 19. Mahida YR. The key role of macrophages in the immunopathogenesis of inflammatory bowel disease. Inflamm Bowel Dis 6: 21–33, 2000. 20. Mayer L. Review article: local and systemic regulation of mucosal immunity. Aliment Pharmacol Ther 11, Suppl 3: 81– 85, 1997. 21. Mayer L. The role of the epithelium in mucosal immunity. Res Immunol 148: 498 –504, 1997. 22. Mester M, Tompkins RG, Gelfand JA, Dinarello CA, Burke JF, and Clark BD. Intestinal production of interleukin-1␣ during endotoxemia in the mouse. J Surg Res 54: 584 –591, 1993. 23. Molmenti EP, Ziambaras T, and Perlmutter DH. Evidence for an acute phase response in human epithelial cells. J Biol Chem 268: 14116 –14126, 1993. 24. Monteleone G, MacDonald TT, Wathen NC, Pallone F, and Pender SL. Enhancing Lamina propria Th1 cell responses with interleukin 12 produces severe tissue injury. Gastroenterology 117: 1069 –1077, 1999. 25. Parlesak A, Schafer C, and Bode C. IgA against gut-derived endotoxins: does it contribute to suppression of hepatic inflammation in alcoholinduced liver disease? Dig Dis Sci 47: 760 –766, 2002. 26. Perez JH, Wight DG, Wyatt JI, Van Schaik M, Mullock BM, and Luzio JP. The polymeric immunoglobulin A receptor is present on hepatocytes in human liver. Immunology 68: 474 – 478, 1989. 27. Pritts T, Hungness E, Wang Q, Robb B, Hershko D, and Hasselgren PO. Mucosal and enterocyte IL-6 production during sepsis and endotoxemia–role of transcription factors and regulation by the stress response. Am J Surg 183: 372–383, 2002. 28. Shimada S, Kawaguchi-Miyashita M, Kushiro A, Sato T, Nanno M, Sako T, Matsuoka Y, Sudo K, Tagawa Y, Iwakura Y, and Ohwaki M. Generation of polymeric immunoglobulin receptor-deficient mouse with marked reduction of secretory IgA. J Immunol 163: 5367–5373, 1999. 29. Sivakumar PV, Westrich GM, Kanaly S, Garka K, Born TL, Derry JM, and Viney JL. Interleukin 18 is a primary mediator of the inflammation associated with dextran sulphate sodium induced colitis: blocking interleukin 18 attenuates intestinal damage. Gut 50: 812– 820, 2002. 30. Spaeth G, Berg RD, Specian RD, and Deitch EA.Food without fiber promotes bacterial translocation from the gut. Surgery 108; 240 –247, 1990. 31. Svanborg C. Bacterial adherence and mucosal immunity. In: Handbook of Mucosal Immunology, edited by Ogra PL, Lamm ME, and McGhee JR. San Diego, CA: Academic, 1974, p. 71–78. 32. Taub DD. Chemokine-leukocyte interactions. The voodoo that they do so well. Cytokine Growth Factor Rev 7: 355–376, 1996. 33. Tliba O, Sibille P, Boulard C, and Chauvin A.Early hepatic cytokine mRNA expression in experimental rat fasciolosis. Vet Parasitol 103; 237–249, 2002. 34. Wang H, Dudley AW Jr, Dupont J, Reeds PJ, Hachey DL, and Dudley MA. The duration of medium-chain triglyceride feeding determines brush border membrane lipid composition and hydrolase activity in newly weaned rats. J Nutr 126: 1455–1462, 1996.

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Clinical implications. It is proposed that medium-chain triglycerides prevent organ injuries and mortality by increasing expression of intestinal secretion of IgA and modulating the intestinal inflammatory immune response in endotoxemia. Thus protection of multiple organs during endotoxemia via an immunological link between the gastrointestinal tract and other organs, such as the liver and lung, could be achieved by immunonutrition using medium-chain triglycerides. Because only one type of medium-chain triglyceride was used, further studies should be performed to determine whether other types of medium-chain triglycerides could be used for clinical purposes. Furthermore, in this study, we concluded that dietary medium-chain triglycerides improved intestinal injury by inhibition of the expression of inflammatory cytokines and chemokines and enhancing intestinal secretory IgA. However, other laboratories have reported that medium-chain triglycerides prevented intestinal atrophy in septic rat (9) and affected the fatty acid composition of the brush-border membrane (34). Thus the direct cytoprotective effects of medium-chain triglycerides on the intestinal epithelial cells could not be proved in this study. Accordingly, further investigation is needed to clarify this issue.

MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY 35. Wang Q, Fischer JE, and Hasselgren PO. Treatment of endotoxemic mice with anti-interleukin-6 antibody paradoxically increases interleukin-6 levels and stimulates mucosal protein synthesis. Arch Surg 132: 82– 88, 1997. 36. Wang Q, Meyer TA, Boyce ST, Wang JJ, Sun X, Tiao G, Fischer JE, and Hasselgren PO. Endotoxemia in mice stimulates production of complement C3 and serum amyloid A in mucosa of small intestine. Am J Physiol Regul Integr Comp Physiol 275: R1584 –R1592, 1998.

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37. Wang Q, Wang JJ, Fischer JE, and Hasselgren PO. Mucosal production of complement C3 and serum amyloid A is differentially regulated in different parts of the gastrointestinal tract during endotoxemia in mice. J Gastrointest Surg 2: 537–546, 1998. 38. Zamir O, Hasselgren PO, Higashiguchi T, Frederick JA, and Fischer JE. Effect of sepsis or cytokine administration on release of gut peptides. Am J Surg 163: 181–184, 1992.

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