Peyer's Patch Lymphocytes Research Articles

Galacto‐Oligosaccharides Modulate Gastrointestinal and Systemic Lymphocyte Populations of SMAD3 Knockout Mice

Galacto‐oligosaccharides (GOS) are prebiotics that support the growth of bifidobacteria and lactobacilli. During influenza vaccination, mice supplemented with GOS had increased T helper (Th) 1 delayed‐type hypersensitivity biomarkers. The objective of our study was to determine the effects of GOS on gastrointestinal and systemic immune cell populations. We hypothesized that GOS would support Th1 responses by increasing CD8+ T cell and Natural Killer (NK) cell numbers. We gavaged 129/Sv SMAD3 knockout mice with 5,000mg GOS/Kg body weight/day for 7 and 14 days and excised blood, spleen, mesenteric lymph nodes (mLN), and Peyer's patches (PPs). Lymphocytes were isolated and stained for flow cytometric analysis of Th cells (CD3+/CD4+), cytotoxic T cells (CD3+/CD8+), B cells (CD19+), and immature (NKp46+/DX5−) and mature NK cells (NKp46+/DX5+). GOS treatment increased total CD4+ T cells in the PPs and mLN within 1 week. GOS also increased CD8+ cytotoxic T lymphocytes in PPs by 1 week and blood by 2 weeks. Mature NK cells were increased in the blood after 2 weeks of GOS treatment. Therefore, GOS treatment modulates both gastrointestinal and systemic immune cell populations. The immune response is Th1 biased during steady‐state conditions in SMAD3 knockout mice treated with GOS. Therefore, GOS may be useful prophylactics and therapeutics. This study was generously supported by GTC Nutritionals.

Adenoviral Transduction of Enterocytes and M-Cells Using in Vitro Models Based on Caco-2 Cells: The Coxsackievirus and Adenovirus Receptor (CAR) Mediates Both Apical and Basolateral Transduction

Understanding virus-cell interaction is a key to the design of successful gene delivery vectors. In the present study we investigated Ad5 transduction of enterocytes and M-cells utilizing differentiated Caco-2 cells and cocultures of Caco-2 cells with lymphocytes. Transduction inhibition studies showed that CAR is the major receptor mediating apical and basolateral virus entry in differentiated Caco-2 cells. Integrins and heparan sulfate glycosaminoglycans do not appear to play a significant role. Immunofluorescence localized CAR to sites of cell-cell contact, with staining mostly observed on the cell perimeter. Staining was observed even in nonpermeabilized monolayers, suggesting apical accessibility of the receptor. Cocultures with mouse Peyer's patch lymphocytes or Raji B human lymphocytes were more susceptible to transduction than Caco-2 cells, and the effects were dose-dependent. Similar to Caco-2 cells, CAR and not integrins mediated apical transduction. In conclusion, contrary to other epithelial cell lines, both apical and basolateral transduction of absorptive enterocytes and M-cells is mediated by binding to CAR. The coculture system can be used to study the interactions between M-cells and gene delivery vectors.

Comparison of the Enteric Mucosal Immunomodulatory Activity of Combinations ofCoptis chinensisFranch. Rhizomes andEvodia rutaecarpa(Juss.) Benth. Fruits in Mice with Dextran Sulphate Sodium-Induced Ulcerative Colitis

Combinations of crude rhizomes of Coptis chinensis Franch., Ranunculaceae, and fruits of Evodia rutaecarpa (Juss.) Benth., Rutaceae, at a ratio of 6 : 1 (formula A) and 1 : 6 (formula B) were extracted with boiling water, and their modulatory activity on enteric mucosal immune responses in mice with dextran sulphate sodium-induced ulcerative colitis was investigated. The results showed that both formulas could reduce the severity of inflammation in the colon. Formula A at a low dose can decrease myeloperoxidase (MPO) activity, and formula B was inactive. Both formulas did not affect the percentages of CD4 (+) and CD8 (+) T cells in the periphery, but they evoked an increase of CD8 (+) T cells among the enteric intraepithelial lymphocytes. Formula B at a low dose could increase both CD4 (+) and CD8 (+) cells, and formula A at a high dose could only increase CD8 (+) T cells among the Peyer's patch lymphocytes (p < 0.05). Both formulas did not affect the percentages of CD4 (+) and CD8 (+) T cells among the lamina propria lymphocytes, but decreased the serum concentration of IL-1 beta (p < 0.05 at a low dose of formula A) and enhanced the level of IL-10 in serum (p < 0.05 at a low dose formula B). We conclude that both formulas have a similar modulating effect on enteric mucosal immune responses, the major difference being that formula A could decrease the level of IL-1 beta, while formula B could increase the IL-10 dose in serum.

Enteric Mucosal Immune Response might Trigger the Immunomodulation Activity ofGanoderma lucidumPolysaccharide in Mice

Ganoderma lucidum is a well-known traditional Chinese medicine and often used for improving quality of life. Ganoderma lucidum polysaccharide (GLP) is the main active component in the fungus. Recent researches have shown that oral administration of GLP could produce immunomodulation and antitumor activity. With a high molecular weight, GLP is not easy to be absorbed, and the pathway via enteric mucosal immune response might be important for the immunomodulation of GLP. To investigate the potential mechanism, Kunming mice, weighing 20 +/- 2 g, were used and their peripheral blood mononuclear cells (PBMC), intraepithelial lymphocytes (IEL) and Peyer's patches lymphocytes (PPL) were separated, and incubated with GLP at different dosages (250 microg/mL, 125 microg/mL, 62.5 microg/mL, 31.25 microg/mL). MTT, ELISA and RT-PCR were used to detect the effects of GLP on the proliferation of the lymphocytes, and expression of IL-10, IL-2 and TNF-alpha. The results showed that GLP could stimulate the proliferation of PBMC and enteric mucosal lymphocytes, and promote the production of IL-2 and IL-10. The RT-PCR results also showed a higher expression of TNF-alpha and IL-10 mRNA in the lymphocytes. The higher expression of IL-2, IL-10 and TNF-alpha from the lymphocytes was observed at GLP concentrations of 62.5 microg/mL and 125 microg/mL. In conclusion, enteric mucosal immune responses could be one of the important pathways for the immunomodulation activity of GLP.

BacteroidesInduce Higher IgA Production ThanLactobacillusby Increasing Activation-Induced Cytidine Deaminase Expression in B Cells in Murine Peyer’s Patches

The gut mucosal immune system is crucial in host defense against infection by pathogenic microbacteria and viruses via the production of IgA. Previous studies have shown that intestinal commensal bacteria enhance mucosal IgA production. However, it is poorly understood how these bacteria induce IgA production and which genera of intestinal commensal bacteria induce IgA production effectively. In this study, we compared the immunomodulatory effects of Bacteroides and Lactobacillus on IgA production by Peyer's patches lymphocytes. IgA production by Peyer's patches lymphocytes co-cultured with Bacteroides was higher than with Lactobacillus. In addition, the expression of activation-induced cytidine deaminase increased in co-culture with Bacteroides but not with Lactobacillus. We found that intestinal commensal bacteria elicited IgA production. In particular, Bacteroides induced the differentiation of Peyer's patches B cell into IgA(+) B cells by increasing activation-induced cytidine deaminase expression.

Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.

Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage

M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.

Reversal of Parenteral Nutrition-induced Gut Mucosal Immunity Impairment With Small Amounts of a Complex Enteral Diet

Although parenteral nutrition (PN) prevents progressive malnutrition, lack of enteral nutrition (EN) during PN leads to gut associated lymphoid tissue (GALT) atrophy and dysfunction. Administering a small amount of EN with PN reportedly prevents increases in intestinal permeability. However, its effects on GALT remain unclear. We analyzed the minimum amount of EN required to preserve gut immunity during PN. Male Institute of Cancer Research mice underwent jugular vein catheter insertion and tube gastrostomy. They were randomized into four groups to receive isocaloric and isonitrogenous nutritional support with variable EN to PN ratios (EN 0, EN 33, EN 66, and EN 100). EN was provided with a complex enteral diet. After 5 days of feeding, the mice were killed and whole small intestines were harvested. GALT lymphocytes were isolated and counted. Their phenotypes were analyzed by flow cytometry. IgA levels of small intestinal washings were analyzed with enzyme-linked immunosorbent assay. Body weight changes did not differ between any two of the groups. Peyer's patch lymphocyte numbers increased in proportion to EN amount, whereas lamina propria lymphocyte numbers were significantly higher in the EN 100 than in the EN 0 group, with no marked increases in the EN 33 and EN 66 groups. Small intestinal IgA levels increased EN amount-dependently and reached a plateau at EN 66. A small amount of EN partially reverses PN-induced GALT changes, suggesting beneficial but limited effects on gut mucosal immunity.

Intestinal immune system of young rats influenced by cocoa-enriched diet

Gut-associated lymphoid tissue (GALT) maintains mucosal homeostasis by counteracting pathogens and inducing a state of nonresponsiveness when it receives signals from food antigens and commensal bacteria. We report for the first time the influence of continuous cocoa consumption on GALT function in rats postweaning. Weaned Wistar rats were fed cocoa-enriched diets (4% or 10% food intake) for 3 weeks. The function of the primary inductive sites of GALT, such as Peyer's patches (PP) and mesenteric lymph nodes (MLN), was evaluated through an analysis of IgA-secretory ability and lymphocyte composition (T, B and natural killer cells), activation (IL-2 secretion and IL-2 receptor α expression) and proliferation. T-helper effector cell balance was also established based on cytokine profile (interferon γ, IL-4 and IL-10) after mitogen activation. A 10% cocoa intake induced significant changes in PP and MLN lymphocyte composition and function, whereas a 4% cocoa diet did not cause significant modifications in either tissues. Cocoa diet strongly reduced secretory IgA (S-IgA) in the intestinal lumen, although IgA's secretory ability was only slightly decreased in PP. In addition, the 10% cocoa diet increased T-cell-antigen receptor γδ cell proportion in both lymphoid tissues. Thus, cocoa intake modulates intestinal immune responses in young rats, influencing γδ T-cells and S-IgA production.

Induction of Preferential Chemotaxis of Unstimulated B‐Lymphocytes by 2‐Arachidonoylglycerol in Immunized Mice

2-Arachidonoylglycerol (2-AG) is an endogenous ligand for cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB2. We investigated the chemotactic activity of 2-AG using mouse lymphocytes because cells in the immune system are known to express CB2 . Spleen cell migration toward 2-AG was observed, which was completely inhibited by SR144528, a CB2-specific antagonist. 2-AG has been reported to induce a preferential B cell chemotaxis. We examined whether there is any difference in responsiveness during the activation of B cells. When spleen cells from immunized mice were tested, naive B cells but not germinal center B cells (GL7-positive) were increased in the fraction attracted by 2-AG. Furthermore, when Peyer's patch lymphocytes were tested after oral administration of cholera toxin, the number of IgA* B cells was increased in the fraction attracted by 2-AG. These results suggested that 2-AG preferentially attracts unstimulated naive B cells rather than activated and/or class-switched B cells. This property may influence the structure of B cell compartments in secondary lymphoid tissues.

Effects of Lymphotoxin β Receptor Blockade on Intestinal Mucosal Immunity

Mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) directs lymphocyte migration into gut-associated lymphoid tissue (GALT) through Peyer's patches (PPs). Parenteral nutrition (PN) impairs mucosal immunity by reducing PPs MAdCAM-1 expression, T and B cells in GALT, and intestinal and respiratory immunoglobulin (Ig) A levels. We previously showed that PN reduces lymphotoxin beta receptor blockade (LTbetaR) in PPs and intestine, and that stimulation with LTbetaR agonist antibodies reverses these defects. To confirm that LTbetaR regulates transcription of MAdCAM-1 message and more fully understand the effects of LTbetaR on MAdCAM-1 function within the mucosal immune system, we studied the effect of LTbetaR blockade with a chimeric LTbetaR Ig-fusion protein on MAdCAM-1 mRNA levels, PP lymphocyte mass and IgA levels in the intestinal and respiratory tracts. Mice were cannulated and killed 3 days after receiving chow + control Ig, chow + LTbetaR-Ig fusion protein (100 microg IV), or PN + control Ig. The PPs of half of the animals were processed for lymphocyte count, and the other half were processed for complementary DNA and subsequent polymerase chain reaction (PCR). mRNA levels of MAdCAM-1 were determined by real-time PCR; intestinal and respiratory IgA levels were measured by ELISA. PN significantly reduced PP lymphocyte mass, MAdCAM-1 mRNA, and intestinal IgA. As anticipated, LTbetaR blockade significantly decreased PP cells and MAdCAM-1 mRNA, but not intestinal IgA because chow feeding was maintained. Both LTbetaR blockade and PN decreased nasal IgA, but not significantly. LTbetaR blockade in chow animals significantly reduces transcription of MAdCAM-1 gene and PPs lymphocyte mass. These data implicate inadequate LTbetaR signaling as a major mechanism for decreased GALT cells with lack of enteral stimulation, and further establish the role of LTbetaR in the mucosal immune system.

LACK OF ENTERAL NUTRITION BLUNTS EXTRACELLULAR-REGULATED KINASE PHOSPHORYLATION IN GUT-ASSOCIATED LYMPHOID TISSUE

The mitogen-activated protein kinase (MAPK) family (extracellular-regulated kinase [ERK], p38, etc.) of signal transduction proteins includes important intracellular mediators of inflammation, playing critical roles in host defense. Phosphorylations of ERK and p38 are responsible for cell proliferation, cell differentiation, and cell death. We hypothesized that impaired gut-associated lymphoid tissue (GALT) function in the absence of enteral nutrition is associated with reduced MAPK phosphorylation in GALT cells. Fifty-three male Institute of Cancer Research mice were randomized into 3 groups; ad libitum chow, intragastric (i.g.)-TPN, and intravenous (i.v.)-TPN. TPN groups were administered a standard TPN solution. After 5 days of feeding, lymphocytes from Peyer patches (PPs), the lamina propria (LP) cells, and intraepithelial (IE) spaces in the small intestine were isolated. GALT lymphocyte numbers were determined. The lymphocytes were incubated with or without 50 ng/mL of phorbol myristate acetate (PMA) for 15 min, and phosphorylated ERK (p-ERK) and p38 (p-p38) levels were determined using laser scanning cytometry. In PP (GALT inductive site) lymphocytes, p-ERK was increased after PMA in all three groups. However, ERK phosphorylation in GALT effector sites (IE and LP) was enhanced only in the enteral groups. p38 phosphorylation was not increased in any GALT sites, in any of the three groups, in response to PMA. In another set of mice (n = 33), in vitro LP lymphocyte proliferation was assessed with BrdU with or without PMA. Cell proliferation was increased or maintained at high level with PMA in the i.g.-TPN and chow group, but remained low in the i.v.-TPN group. In conclusion, lack of enteral feeding blunts ERK activation and cell proliferation in response to PMA stimulation in GALT effector sites, which may be an important mechanism underlying reduced GALT function. The influence of nutrition on GALT p38 phosphorylation must be assessed with other types and dosages of stimulants.

Effects of Ginseng polysaccharides and polyporus polysaccharides on TNF-α and IFN-γ-production by enteric mucosal lymphocytes of rats with collagen induced arthritis

The effects of Ginseng and Polyporus polysaccharides (from Ginseng and Polyporus umbellatus (Pers) Fries) [1,2] on the TNF-α and IFN-γ-production by enteric mucosal lymphocytes in collagen induced arthritis (CIA) rats were investigated. The Peyer's patches lymphocytes (PPL), intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of SD rats were isolated, and checked with flow cytometry after co-culturing with Ginseng and Polyporus polysaccharides at different dosages. ELISA was used to measure the levels of TNF-α and IFN-γ in the supernatants. The arthritic incidence of CIA in SD rats was 17/20at the week 4 after the immunization. More CD4 cells and more CD8 cells were detected in PPL, IEL and LPL in CIA models. Gingseng polysaccharides at 0.15g/L, 0.30g/L and 0.60g/L can significantly lower the secretion of TNF-α in PPL of CIA rats. At 0.15g/L and 0.30g/L, it can lower the secretion of TNF-α in IEL of CIA rats, and it can lower the secretion of TNF-α in LPL of CIA rats. Polyporus polysaccharides at 1.0g/L and 2.0g/L can significantly lower the secretion of TNF-α in PPL of CIA rat and the secretion of TNF-α in IEL of CIA rats. At 1.0g/L and 2.0g/L, it can lower the secretion of TNF-α in LPL of CIA rats. Ginseng polysaccharides at 0.15g/L, 0.30g/L and 0.60g/L can significantly lower the secretion of IFN-γin IEL and LPL of CIA rats, and increase the secretion of IFN-γin PPL of CIA rats. Polyporus polysaccharides at 0.5g/L, 1.0g/L and 2.0g/L can significantly lower the secretion of IFN-γin PPL, IEL and LPL of CIA rat, and increase the secretion of IFN-γin PPL of CIA rats. Obvious dose dependent responses in PPL were found in ginseng and polyporus polysaccharides. The results indicate that Ginseng and Polyporus polysaccharides could affect the enteric mucosal immune response in CIA rats.

Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum

In order to determine the role of Peyeros patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.

An in vitro cell-culture model demonstrates internalin- and hemolysin-independent translocation of Listeria monocytogenes across M cells

An ability to translocate the mucosal epithelia through M cells provides invasive pathogens with a rapid means of accessing the mucosal lymphoid tissues. In order to determine the role of M cells in Listeria monocytogenes infection, we initially assessed colonization of Peyer's patch (PP) epithelium in BALB/c mice by Vibrio cholerae Eltor, wild-type L. monocytogenes and an isogenic hemolysin mutant (LO28Δ hly). It was observed that both wild-type L. monocytogenes and Δ hly showed preferential colonization of PP epithelium in this model. Furthermore, a novel luciferase reporter system was used to show rapid site-specific localization of L. monocytogenes in intestinal Peyer's patches. To examine the role of M cells in transcytosis of L. monocytogenes we utilized an in vitro transwell model that mimics M-cell activity through differentiation of C2Bbe1 epithelial enterocytes via co-culture with murine Peyer's patch lymphocytes (PPL). It was shown that L. monocytogenes transits M cells at significantly increased rates compared to C2Bbe1 monocultures. In addition, M-cell transport occurred independently of bacterial hemolysin and internalin production. This study demonstrates rapid transcytosis of L. monocytogenes through M cells, a process that occurs independently of the action of classical virulence factors.

Influences of Long‐Term Antibiotic Administration on Peyer's Patch Lymphocytes and Mucosal Immunoglobulin A Levels in a Mouse Model

Long-term antibiotic administration is sometimes necessary to control bacterial infections during the perioperative period. However, antibiotic administration may alter gut bacterial flora, possibly impairing gut mucosal immunity. We hypothesized that 1 week of subcutaneous (SC) antibiotic injections would affect Peyer's patch (PP) lymphocyte numbers and phenotypes, as well as mucosal immunoglobulin A (IgA) levels. Sixty-one male Institute of Cancer Research mice were randomized to CMZ (cefmetazole 100 mg/kg, administered SC twice a day), IPM (imipenem/cilastatin 50 mg/kg x 2), and control (saline 0.1 mL x 2) groups. After 7 days of treatment, the mice were killed and their small intestines removed. Bacterial numbers in the small intestine were determined using sheep blood agar plates under aerobic conditions (n = 21). PP lymphocytes were isolated to determine cell numbers and phenotypes (CD4, CD8, alphabetaTCR, gammadeltaTCR, B220; n = 40). IgA levels in the small intestinal and bronchoalveolar washings were also measured with ELISA. Antibiotic administration decreased both bacterial number and the PP cell yield compared with the control group. There were no significant differences in either phenotype percentages or IgA levels at any mucosal sites among the 3 groups. Long-term antibiotic treatment reduces PP cell numbers while decreasing bacterial numbers in the small intestine. It may be important to recognize changes in gut mucosal immunity during long-term antibiotic administration.

Parenteral Nutrition Impairs Gut-Associated Lymphoid Tissue and Mucosal Immunity by Reducing Lymphotoxin β Receptor Expression

To determine the effects of parenteral nutrition (PN) on LTbetaR in gut-associated lymphoid tissue (GALT), particularly the intestine and Peyer's patches (PP). Lack of enteral stimulation with PN impairs mucosal immunity and reduces IgA levels through depression of GALT cytokines (IL-4 and IL-10) and GALT specific adhesion molecules. We have shown that each is critical to intact mucosal immunity through effects on lymphocyte homing, IgA production, and resistance to antibacterial and antiviral immunity. IgA is the principal specific immunologic mucosal defense. LTbetaR stimulation controls production of IL-4, the adhesion molecule MAdCAM-1, and other key components of GALT, all of which are important in increasing IgA levels and maintaining mucosal defenses. Experiment 1: LTbetaR expression in intestine and PP was analyzed by Western blot after 5 days of chow, a complex enteral diet (CED), or PN. Diets were isocaloric and isonitrogenous except for chow. Experiment 2: After completing pilot experiments to determine the appropriate dose of the LTbetaR agonistic antibody, mice received chow, PN + 5 mug of anti-LTbetaR mAb (2 times/d, i.v.) or PN + isotype control antibody. PP lymphocytes and intestinal IgA levels were measured after 2 days. Lack of enteral stimulation with PN significantly decreased LTbetaR expression in intestine and PP compared with chow and CED. LTbetaR stimulation with an agonistic anti-LTbetaR mAb significantly increased PP lymphocyte counts and intestinal IgA in PN fed-mice. LTbetaR expression is critical for GALT control mechanisms and intact mucosal immunity. PN reduces LTbetaR expression, PP lymphocytes, and intestinal IgA production. Exogenous LTbetaR stimulation reverses PN-induced depression of gut mucosal immunity.

Impairment of lymphocyte function in head-injured rats: Effects of standard and immune-enhancing diets for enteral nutrition

The metabolic response to head injury (HI) is characterized by a dysimmunity which may be a risk factor of a septic state. The use of immune enhancing diets (IEDs) could be a promising approach to improve immune functions. The aim of the study was to investigate the consequences of HI on lymphocyte function and to determine the effects of an enteral IED comparatively to a standard enteral nutrition. A rat model of HI by fluid percussion was used. Twenty-five male Sprague-Dawley rats were randomized into 4 groups: rats receiving standard chow diet ad libitum (AL), rats sustaining HI and receiving standard chow diet and enteral saline (HI), rats receiving the enteral standard diet Sondalis HP (HIS), and rats receiving the IED Crucial (HIC). The two enteral diets were infused continuously during 4 days after the HI and were isocaloric, isonitrogenous and isovolumic. HI induced a thymus atrophy (HI vs. AL, P<0.05), and an impairment in lymphocyte CD25 receptor density responsiveness to stimulation. The IED blunted thymus atrophy and allowed to preserve the stimulation of blood and Peyer patches lymphocytes (HIC: Stimulated vs. Basal, P<0.05). IED seems more adapted for preserving lymphocyte function than standard diet in HI patients.

Multipotential acceptance of Peyer's patches in the intestine for both thymus‐derived T cells and extrathymic T cells in mice

Peyer's patches (PP) are important inductive sites for the mucosal immune response. It is well known that lymphocytes that migrate into PP are mainly of T-cell lineage from thymus-derived cells (i.e. alphabetaTCR(high) cells). In this study, we further characterized the properties of PP lymphocytes in mice using a mouse model of colitis induced by dextran sulphate sodium (DSS). Although the major site of the inflammation induced by DSS is known to be the large intestine, the small intestine was also damaged. When mice developed DSS-induced colitis, CD3+CD8+B220+ gammadelta T cells increased in PP in the small intestine. These gammadelta T cells, which are not seen in the PP of normal mice, resembled intraepithelial lymphocytes (IEL) in the small intestine in terms of their expression of CD5, CD103 and Thy1.2. In addition, the Vgamma/delta repertoire of these gammadelta T cells was similar to that of gammadelta IEL. When DSS-treated mice were injected with IEL isolated from normal mice, IEL including gammadelta T cells preferentially migrated to PP, raising the possibility that B220+ T cells seen in PP of diseased mice may derive from IEL in the small intestine. Our present study suggests that PP might be able to accept T-cell lineages from intestinal IEL as well as from thymus-derived T cells.

Absence of CCR6 Inhibits CD4 + Regulatory T-Cell Development and M-Cell Formation inside Peyer's Patches

The chemokine Mip3alpha is specifically expressed by the follicle-associated epithelia (FAE) covering intestinal Peyer's patches (PPs) and is the only known chemokine ligand for the chemokine receptor CCR6. Although CCR6-deficient mice are known to have a perturbed intestinal immune system, little is known about the specific impact of this interaction for Peyer's patch formation. To elucidate the effect of Mip3alpha on PP lymphocyte development, we used a CCR6/enhanced green fluorescent protein (EGFP) knock-in mouse model and analyzed lymphocyte development by immunohistochemistry and flow cytometry. PPs of CCR6-/- mice were significantly size-reduced with a proportional loss of B cells and T cells, whereas T-cell subsets were disturbed with a decreased CD4/CD8 ratio paralleled with a loss of regulatory CD4+ CD45Rb(low) T cells. The analysis of cytokine production by CCR6-expressing cells could demonstrate that CCR6 is involved in the regulation of cytokine secretion such as interleukin-12 by dendritic cells. Quantification of UEA-1+ cells inside the FAE showed reduced M-cell numbers in CCR6-deficient mice. These results suggest that the interaction of CCR6 with its ligand Mip3alpha is important for immune responses generated inside the PPs, particularly for the generation of regulatory CD4+ T cells residing inside PPs and for the formation of M cells.