This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Kang, W. Articles by Maeshima, Y. Search for Related Content PubMed PubMed Citation Articles by Kang, W. Articles by Maeshima, Y. Pubmed/NCBI databases Substance via MeSH Social Bookmarking                   What's this?

Effects of Lymphotoxin β Receptor Blockade on Intestinal Mucosal Immunity

Woodae Kang, MD, PhD, Kenneth A. Kudsk, MD^*,, Yoshifumi Sano, MD, Jinggang Lan, PhD, Fu Yang-Xin, PhD, F. Enrique Gomez, PhD and Yoshinori Maeshima, MD

From the ^* Veterans Administration Surgical Services, William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin; the Department of Surgery, University of Wisconsin–Madison College of Medicine and Public Health, Madison, Wisconsin; and University of Chicago Department of Pathology, Chicago, Illinois

Correspondence: Kenneth A. Kudsk, MD, 600 Highland Ave, H4/736 CSC, Madison, WI 53792-7375. Electronic mail may be sent to kudsk{at}surgery.wisc.edu.

Background: 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 β receptor blockade (LTβR) in PPs and intestine, and that stimulation with LTβR agonist antibodies reverses these defects. To confirm that LTβR regulates transcription of MAdCAM-1 message and more fully understand the effects of LTβR on MAdCAM-1 function within the mucosal immune system, we studied the effect of LTβR blockade with a chimeric LTβR Ig-fusion protein on MAdCAM-1 mRNA levels, PP lymphocyte mass and IgA levels in the intestinal and respiratory tracts. Methods: Mice were cannulated and killed 3 days after receiving chow + control Ig, chow + LTβR-Ig fusion protein (100 µg 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. Results: PN significantly reduced PP lymphocyte mass, MAdCAM-1 mRNA, and intestinal IgA. As anticipated, LTβR blockade significantly decreased PP cells and MAdCAM-1 mRNA, but not intestinal IgA because chow feeding was maintained. Both LTβR blockade and PN decreased nasal IgA, but not significantly. Conclusions: LTβR blockade in chow animals significantly reduces transcription of MAdCAM-1 gene and PPs lymphocyte mass. These data implicate inadequate LTβR signaling as a major mechanism for decreased GALT cells with lack of enteral stimulation, and further establish the role of LTβR in the mucosal immune system.

Parenteral nutrition (PN) with lack of enteral nutrition increases the incidence of pneumonia and intraabdominal abscesses in critically injured patients.^1,2 According to the common mucosal immune system hypothesis, the Peyer's patches (PPs) of the gut-associated lymphoid tissue (GALT) are considered the major inductive site for initiation of antigen-specific immunoglobulin (Ig) A production. Naïve T and B cells migrate from the circulation into PPs, are sensitized to antigens processed in the PPs, and are distributed via the systemic circulation to the lamina propria of intestinal and extraintestinal (such as the respiratory tract) mucosal sites after passing through mesenteric lymph nodes and the thoracic duct.³ In these lamina propria effector sites, the sensitized T and B cells cooperate to produce antigen-specific dimeric IgA, which serves as the antigen-specific immune defense at mucosal surfaces in the form of secretory IgA.⁴

PN with lack of enteral nutrition reduces the absolute numbers of intraepithelial and lamina propria lymphocytes and PPs,⁵ decreases the levels of interleukin (IL)-4 and IL-6 (2 important Th2-type IgA-stimulating cytokines) in the lamina propria,⁶ and reduces intestinal and respiratory tract IgA levels.⁷ The overall effect of these changes destroys both antiviral and antibacterial respiratory defenses.^8,9 These effects can be traced back to initial effects of PN on mucosal addressin cellular adhesion molecule-1 (MAdCAM-1). MAdCAM-1, expressed on the high endothelial venules of PPs, is a key molecule that regulates the traffic of lymphocytes into the mucosal immune system.^10,11 This molecule, together with chemokines produced locally, draws naïve T and B cells into the PPs to start the process of mucosal immune protection. Our prior work shows that the PN-induced GALT cell mass reductions initially derive from decreased MAdCAM-1 mRNA and protein expression, reducing entry of T and B cells into the PPs.¹² This effect is reproducible by blocking MAdCAM-1 with a specific anti-MAdCAM-1 antibody.¹³

Lymphotoxin β receptor (LTβR) signaling plays a critical role in PPs' organogenesis and regulation of both MAdCAM-1 expression and IL-4 production^14–16 and it induces CCL20 chemokine expression through NFB in gut mucosal cells.¹⁷ Chemokines are molecules that stimulate the migration of cells into tissues.¹⁸ LTβR is expressed on the surface of cells in the parenchyma and stroma of most lymphoid organs but is conspicuously absent on T and B lymphocytes. One of the ligands for LTβR is lymphotoxin (LT1β2), which is transiently expressed on the surface of activated T and B lymphocytes. Receptor ligation leads to activation of 2 distinct forms of NFB and expression of genes involved in regulating immune processes that contribute to cell survival, cell migration, and other events.¹⁵ We previously showed that PN decreases LTβR expression in the PPs and intestine and that administration of a stimulatory (agonistic) antibody to LTβR reversed the PN-induced decrease in PPs lymphocyte count and intestinal IgA.¹⁹ The purpose of this study was to describe the relationship between LTβR signaling with the PN-induced impaired mucosal immunity by specifically blocking LTβR to further our investigation of the mechanism of PN-induced MAdCAM-1 changes. For this reason, we examined effects of LTβR blockade on PPs lymphocyte count, intestinal and upper respiratory IgA levels, PPs MAdCAM-1 gene expression, and the gene expression of 2 chemokines (CCL20 and CXCL12) important in cell migration in chow-fed mice and compared them to PN feeding.

	MATERIALS AND METHODS

Top MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY

 
Animals
All experimental protocols were approved by the Animal Care and Use Committee of the University of Wisconsin–Madison and Middleton Veterans Administration Hospital, Madison. Six-week-old, male, Institute of Cancer Research mice (Harlan, Indianapolis, IN) were used for the experiments. The mice were kept in an environment under controlled temperature, humidity, and light cycle (12-hour light/12-hour dark). They were acclimatized for 1 week and fed a standard mouse chow (PMI Nutrition International, St. Louis, MO) ad libitum before protocol entry. During feeding protocol, mice had free access to water and were individually housed in metal metabolism cages with wire grid floors to eliminate coprophagia.

Feeding and Experimental Protocol
Forty-three mice received central venous catheters (0.3-mm ID and 0.6-mm OD; Helix, Carpinteria, CA) under ketamine/acepromazine (100/10 mg/kg) anesthesia as described in detail previously.^5–7 Catheterized mice were placed into metal metabolism cages and immediately connected to infusion pumps. This procedure is an acceptable method of nutrition support that does not induce physical and biochemical stress.²⁰ The mice were given 0.9% saline at 4 mL/d for 48 hours through the catheter, with free access to chow. After recovering from surgery for 2 days, the animals were then randomly assigned to receive either 100 µg LTβR-Ig fusion protein IV (LTβR-Ig, 100 µg in 250 µL PBS; a generous gift from Yang-Xin Fu, Chicago University, Chicago, IL) or human IgG IV (100 µg in 250 µL PBS; Sigma, St. Louis, MO) as control. The LTβR-Ig-treated mice continued with chow ad libitum and 0.9% saline at 4 mL/d IV throughout the study. The control Ig-treated mice were randomly assigned to either chow or IV PN. The PN mice initially received 4 mL/d of PN and were advanced to a goal rate of 10 mL/d by the third day of feeding. The PN solution contained 6.0% amino acids, 34.9% dextrose (6002 kJ/L), electrolytes, and multivitamins, with a nonprotein calorie/nitrogen ratio of 535.8 kJ/g nitrogen. After 3 days of these dietary treatments, the animals were anesthetized with a ketamine/acepromazine mixture and exsanguinated by cardiac puncture. The small intestine was excised from the pylorus to terminal ileum and the mesenteric fat and external vasculature were dissected away. Then, the intestinal lumen was flushed by 20 mL calcium- and magnesium-free Hanks' balanced salt solution (HBSS). Nasal washes were collected by 1-mL saline injection through an 18-gauge angiocath inserted into the tracheal lumen at the level of the cricoid cartilage after a midline incision made over the ventral aspect of the trachea slightly superior to the thoracic inlet; these washing fluids were collected in plastic tubes and stored at –80°C. The PPs were also removed from the intestine either for lymphocyte cell count (see below) or frozen in liquid N₂ and stored at –80°C for analysis of mRNA by real-time polymerase chain reaction (PCR).

Cell Isolation
Lymphocyte isolation from the PPs was performed following the previously described protocol, with minor modifications.⁵ In brief, the PPs were excised from the serosal side of the intestine and the number was counted. Then, the PPs were teased apart with scissors and the fragments were incubated with RPMI-1640 (Mediatech Inc, Herndon, VA) containing collagenase (Sigma; 40 U/mL), 5% fetal bovine serum, 100 U/mL penicillin-streptomycin, and 2 mmol/L D-glutamine for 1 h at 37°C with constant rocking. The tissue slurry was passed through a 100-µm nylon cell strainer and centrifuged at 1500 rpm for 5 minutes. The cell pellet was resuspended in RPMI-1640 solution without collagenase. PP lymphocyte number was determined by counting in a hemocytometer after trypan blue staining.

IgA Quantification
IgA was measured in intestinal and nasal washes with a sandwich ELISA,¹⁹ with a polyclonal goat antimouse IgA (Sigma) to coat a plate, a myeloma mouse IgA as standard (Sigma), and a peroxidase-conjugated goat antimouse IgA antibody (Sigma).

RNA Isolation and PCR
RNA isolation and quantitation. Total RNA was isolated with TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, the PP samples were homogenized with 1 mL of TRIzol and the RNA was extracted with chloroform and precipitated with isopropanol. The integrity of the RNA was checked with 1% agarose gels stained with ethidium bromide by visualization of the 28S and 18S rRNA bands; its concentration was determined by UV at 260 nm and stored at –80°C until required.

Reverse transcription. Total RNA was used for the synthesis of single-strand complementary DNA (cDNA) using avian myeloblastoma virus reverse transcriptase (AMV-RT). All reagents used for reverse transcription were from Promega (Madison, WI). Total RNA (2 µg) was incubated for 5 minutes at 70°C with 1 µg of oligo(dT)₁₅, chilled on ice for 5 minutes, and further incubated for 1 hour at 42°C with 2.5 µL of a 40 mmol/L deoxynucleotide triphosphates (dNTPs), 30 U of AMV-RT, 5 µL of 5x AMV-RT buffer, and 40 U of ribonuclease inhibitor (RNasin) in a final volume of 25 µL. Reactions were then heat inactivated at 70°C for 10 minutes and the cDNA was stored at –20°C until analysis.

Gene expression by quantitative real-time PCR. Real-time PCR was performed using a Rotor-Gene 3000 thermal cycling system (Corbett Research, Sydney, Australia) with SYBR-Green I (Molecular Probes, Eugene, OR) as fluorescent probe. Briefly, PCR reactions were performed with 2 µL of 1:10 diluted cDNA, 0.3 µmol/L each of forward and reverse primers (Table I), and a cocktail mixture containing MgCl₂, SYBR-Green, and Platinum Quantitative PCR Super-Mix-UDG (Invitrogen, Life Technologies) in a total volume of 10 µL. The reaction mixture was preheated 2 minutes at 50°C and 2 minutes at 95°C; then heated for 45 cycles with 15 seconds at 95°C, 20 seconds at 58°C, 15 seconds at 72°C, and 15 seconds at 78°C; and followed by melting curve analysis to establish product specificity. A standard curve was constructed with 6-fold serial dilutions of cDNA from PPs in a chow-fed mouse, and all reaction tubes were run in triplicate. The 6 points always showed a strong linear relationship (R² >0.99) with the threshold cycle. Data from each tube were analyzed using Rotor-Gene 3000 Application Software 6.0 (Corbett Research), and the mRNA levels of the target gene were obtained by plotting on the standard curve. The value of each gene was normalized with the level of 18S rRNA as housekeeping gene.

Statistical Analyses
All values were expressed as mean ± SE. Statistical analysis was performed by ANOVA, followed by the Fisher's protected least significant difference post hoc test.

	RESULTS

Top MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY

 
Body Weight Change
There were no significant differences between groups in preexperiment body weight (Table II). At the end of the feeding protocol, PN-fed mice lost significantly more weight than the chow-fed groups; there were no significant differences between chow + control Ig and chow + LTβR-Ig groups. Chow-fed mice typically have 1.5 g of residual feces, whereas the GI tracts of PN mice are empty. Therefore, the body weight differences were exaggerated between PN and chowfed mice.

Lymphocytes Numbers in PPs
PP total lymphocyte counts were significantly lower in the chow + LTβR-Ig (n = 6) and PN + control Ig (n = 8) mice compared with the chow + control Ig (n = 6) group (Figure 1A). Chow + LTβR-Ig produced significantly higher total cell counts compared with PN + control Ig mice. There were no significant differences in the number of harvested PPs (chow + control Ig: 10.7 ± 0.7; chow + LTβR-Ig: 10.3 ± 0.8; and PN + control Ig: 10.4 ± 0.6). Lymphocyte counts/PP were significantly decreased in the chow + LTβR-Ig (n = 6) and the PN + control Ig (n = 8) mice compared with those in the chow + control Ig (n = 6) group (Figure 1B). There were significantly higher lymphocyte counts/PP in chow + LTβR-Ig mice compared with PN + control Ig mice.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Effects of lymphotoxin β receptor-immunoglobulin fusion protein (LTβR-Ig) and PN on lymphocyte numbers in Peyer's patches (PPs). A, Total lymphocyte numbers in PPs. B, Lymphocyte numbers/PP. *p < .05 vs chow + control Ig. p< .05 vs chow + LTβR-Ig.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Effects of lymphotoxin β receptor-immunoglobulin fusion protein (LTβR-Ig) and PN on gene expression of mucosal addressin cellular molecule-1 (MAdCAM-1). The data are expressed as percent of chow + control Ig group. *p < .05 vs chow + control Ig group.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Effects of lymphotoxin β receptor-immunoglobulin fusion protein (LTβR-Ig) and PN on gene expression of CCL20 and CXCL12. The data are expressed as percent of chow + control Ig group.

IgA Levels
The levels of intestinal IgA were similar in both chow + control Ig (n = 14) and chow + LTβR-Ig (n = 13) groups, whereas those in the PN + control Ig mice (n = 16) were significantly reduced (Figure 4).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Effects of lymphotoxin β receptor-immunoglobulin fusion protein (LTβR-Ig) and PN on intestinal IgA levels. PN significantly reduced intestinal IgA levels. LTβR-Ig administration did not decrease intestinal IgA levels. *p < .05 vs chow + control Ig and chow + LTβR-Ig group.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Effects of lymphotoxin β receptor-immunoglobulin fusion protein (LTβR-Ig) and PN on nasal IgA levels.

	DISCUSSION

Top MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY

 
PPs are a primary sensitization site for naïve T and B lymphocytes destined for the mucosal immune network. After the sensitized lymphocytes mature or proliferate in mesenteric lymph nodes, they enter the thoracic duct, spread via blood circulation, and localize to the intestine or other extraintestinal mucosal effector sites.^4,21 There, the B cells differentiate into plasma cells capable of producing secretory IgA,⁴ which is the major antigen-specific mucosal immune defense against bacteria.

Experimentally, our work with GALT and the mucosal-associated lymphoid tissue (MALT) in mice provides a logical framework to explain the preponderance of clinical data demonstrating reductions in pneumonia when critically injured patients are fed enterally rather than parenterally or not at all.^1,2 In these models, animals fed parenterally, with no enteral stimulation, exhibit impaired mucosal immunity, which is initially reflected in decreased MAdCAM-1 expression in PPs, which ultimately results in reduced GALT cell mass.^12,13 Simultaneously, intestinal and respiratory tract IgA levels drop.^5,7,22 These changes induce functional defects in antiviral and antibacterial respiratory defenses. Animals immune to respiratory challenge with the A/PR (H1N1) influenza virus or Pseudomonas aeruginosa lose established IgA-mediated respiratory immunity after parenteral feeding but not with chow or a complex enteral diet.^8,9 Similarly, PN-fed mice fail to generate new antibody-forming cells in response to an acute viral respiratory infection, which depresses the viral-specific respiratory antibody response.^23–25 These defects are rapidly reversible with chow refeeding.⁷

A unique combination of adhesion molecules and chemokines mediates migration of T and B lymphocytes into PPs from the circulation. Lymphocytes expressing both ₄β₇ and L-selectin on their surface interact with MAdCAM-1 and intercellular adhesion molecule-1 (ICAM-1), which are expressed on the high endothelial venules of the PPs²⁶ and initiate rolling and the adhesion of the cells to the venous endothelium. MAdCAM-1 is key to these interactions because MAdCAM-1 blockade by an antagonistic antibody reduces PP and LP lymphocyte levels to those of PN-fed mice given no enteral stimulation.¹³ ICAM-1 blockade has no effect.²⁶ MAdCAM-1 is expressed in response to LTβR signaling after stimulation by lymphotoxin.^27–29 In this study, we demonstrate that inhibition of LTβR signaling significantly decreases PPs lymphocyte number in toto or per PP, as well as decreases PP MAdCAM-1 gene expression to levels similar to those of PN-fed mice. These results support our hypothesis that lack of enteral stimulation plus PN depresses LTβR expression, resulting in impaired mucosal immunity. This is consistent with our previous data showing that LTβR stimulation with an agonistic antibody reverses the lack of enteral nutrition, with PN-induced depression in PP cell counts.¹⁹

However, blockade with LTβR-Ig did not decrease lymphocytes in PPs to levels of PN-feeding alone, and there are several potential explanations for this observation. It is possible that trafficking-related adhesion molecules other than MAdCAM-1 are reduced by PN but not by LTβR blockade. Because G protein–coupled receptors induce integrin-dependent firm adhesion in lymphocytes trafficking process,¹¹ PN might strongly suppress these proteins, whereas LTβR blockade would not. Second, maximal enteral stimulation with chow produces compensatory effects, as we showed previously in our MAdCAM-1 blockade experiment.¹³ Although MAdCAM-1 blockade reduced cells numbers in GALT, intestinal IgA levels were not suppressed to PN levels. This implies that depressed MAdCAM-1 expression through LTβR signaling inhibition is a major mechanism for the decreased PP lymphocytes' mass with PN, but additional factors other than cell mass influence intestinal IgA levels. Those additional factors include compensatory increases in intestinal IL-4 levels, which we noted in previous work, which could blunt the overall effect of reduced cell mass.³⁰ How chow stimulation maintained the IL-4 levels is unknown, but IL-4 is one of the Th-2 type IgA-stimulating cytokines affected by PN with lack of enteral stimulation. In addition, enteral stimulation induces the release of neuropeptides, including gastrin-releasing peptide, gastrin, and cholecystokinin, which are known to support mucosal immunity.^31–36 Enteral stimulation induces formation of small lymphoid clusters, termed isolated lymphoid follicles (ILF), which are associated with mucosal IgA production in the small intestine.³⁷ Additional effects could be generated by altered bacterial flora.^38,39 Administration of LTβR-Ig reportedly reduces ILF formation induced by oral toxin immunization after a short period of food deprivation,²⁷ whereas continuous oral feeding maintains IgA production in ILF by maintaining normal bacterial flora. PPs are not a prerequisite to generation of IgA-producing cells.⁴⁰ Additionally, lack of luminal nutrients seems to disrupt the transport system of IgA from the basal surface of epithelial cells to the luminal surface.²⁴ Chow-fed animals might be able to maintain any of these systems despite LTβR signal blockade.

This work has some limitations, however. Our primary interest remains how enteral stimulation reduces pneumonia in critically ill patients. Our prior work demonstrated impaired antiviral and antibacterial defenses, presumably due to impaired IgA-mediated mucosal defenses. This work sheds light on gene, GALT cell, and IgA responses to LTβR blockade but does not test alterations in the function of those defenses.

	SUMMARY

Top MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY

 
This work supports the importance of LTβR levels and LTβR's stimulation in the maintenance of normal mucosal immunity. This stimulation is lost under conditions in which there is no enteral stimulation. Taken together with our pervious stimulatory agonist experiments, the key role of this molecule on mucosal immunity in normal conditions is clear.

The study was supported by National Institutes of Health grant R01 GM53439.

Received for publication December 12, 2006. Accepted for publication January 22, 2007.

1. Kudsk KA, Minard G, Croce MA, et al. A randomized trial of isonitrogenous enteral diets following severe trauma: an immune-enhancing diet (IED) reduces septic complications. Ann Surg.1996; 224:531 –543.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
2. Kudsk KA, Croce MA, Fabian TC, et al. Enteral vs. parenteral feeding: effects on septic morbidity following blunt and penetrating abdominal trauma. Ann Surg. 1992;215:503 –513.[Web of Science][Medline] [Order article via Infotrieve]
3. Roux ME, McWilliams M, Phillips-Quagliata JM, Lamm ME. Differentiation pathway of Peyer's patch precursors of IgA plasma cells in the secretory immune system. Cell Immunol.1981; 61:141 –153.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
4. McGhee JR, Mestecky J, Dertzbaugh MT, Eldridge JH, Hirasawa M, Kiyono H. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine.1992; 10:75 –88.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
5. Li J, Kudsk KA, Gocinski B, Dent D, Glezer J, Langkamp-Henken B. Effects of parenteral and enteral nutrition on gut-associated lymphoid tissue.J Trauma. 1995;39:44 –52.[Web of Science][Medline] [Order article via Infotrieve]
6. Wu Y, Kudsk KA, DeWitt RC, Tolley EA, Li J. Route and type of nutrition influence IgA-mediating intestinal cytokines. Ann Surg. 1999;229:662 –667.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
7. King BK, Li J, Kudsk KA. A temporal study of TPN-induced changes in gut-associated lymphoid tissue and mucosal immunity. Arch Surg.1997; 132:1303 –1309.[Abstract/Free Full Text]
8. Kudsk KA, Li J, Renegar KB. Loss of upper respiratory tract immunity with parenteral feeding. Ann Surg.1996; 223:629 –638.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
9. King BK, Kudsk KA, Li J, et al. Route and type of nutrition influence mucosal immunity to bacterial pneumonia. Ann Surg.1999; 229:272 –278.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
10. Briskin M, Winsor-Hines D, Shyjan A, et al. Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue. Am J Pathol.1997; 151:97 –110.[Abstract]
11. Butcher EC, Picker LJ. Lymphocyte homing and homeostasis.Science. 1996;272:60 –67.[Abstract]
12. Gomez FE, Lan J, Kang W, Ueno C, Kudsk K. Parenteral nutrition and fasting reduces mucosal addressin cellular adhesion molecule-1 mRNA in Peyer's patches of mice. JPEN J Parenter Enteral Nutr.2007; 31:1 –6.[Abstract/Free Full Text]
13. Ikeda S, Kudsk KA, Fukatsu K, et al. Enteral feeding preserves mucosal immunity despite in vivo MAdCAM-1 blockade of lymphocyte homing.Ann Surg. 2003;237:677 –685.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
14. Spahn TW, Eugster HP, Fontana A, Domschke W, Kucharzik T. Role of lymphotoxin in experimental models of infectious diseases: potential benefits and risks of a therapeutic inhibition of the lymphotoxin-beta receptor pathway. Infect Immun.2005; 73:7077 –7088.[Free Full Text]
15. Stopfer P, Obermeier F, Dunger N, et al. Blocking lymphotoxin-beta receptor activation diminishes inflammation via reduced mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expression and leucocyte margination in chronic DSS-induced colitis. Clin Exp Immunol.2004; 136:21 –29.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
16. Stopfer P, Mannel DN, Hehlgans T. Lymphotoxin-beta receptor activation by activated T cells induces cytokine release from mouse bone marrow-derived mast cells. J Immunol.2004; 172:7459 –7465.[Abstract/Free Full Text]
17. Rumbo M, Sierro F, Debard N, Kraehenbuhl JP, Finke D. Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.Gastroenterology. 2004;127:213 –223.[CrossRef][Medline] [Order article via Infotrieve]
18. Okada T, Ngo VN, Ekland EH, et al. Chemokine requirements for B cell entry to lymph nodes and Peyer's patches. J Exp Med.2002; 196:65 –75.[Abstract/Free Full Text]
19. Kang W, Gomez FE, Lan J, Sano Y, Ueno C, Kudsk KA. Parenteral nutrition impairs gut-associated lymphoid tissue and mucosal immunity by reducing lymphotoxin beta receptor expression. Ann Surg.2006; 244:392 –399.[Web of Science][Medline] [Order article via Infotrieve]
20. Sitren HS, Heller PA, Bailey LB, et al. Total parenteral nutrition in the mouse: development of a technique. JPEN J Parenter Enteral Nutr. 1983;7:582 –586.[Abstract/Free Full Text]
21. Brandtzaeg P, Pabst R. Let's go mucosal: communication on slippery ground. Trends Immunol.2004; 25:570 –577.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
22. Janu P, Li J, Renegar KB, Kudsk KA. Recovery of gut-associated lymphoid tissue and upper respiratory tract immunity after parenteral nutrition. Ann Surg.1997; 225:707 –715.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
23. Johnson CD, Kudsk KA, Fukatsu K, Renegar KB, Zarzaur BL. Route of nutrition influences generation of antibody-forming cells and initial defense to an active viral infection in the upper respiratory tract. Ann Surg. 2003;237:565 –573.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
24. Renegar KB, Kudsk KA, DeWitt RC, et al. Impairment of mucosal immunity by parenteral nutrition: depressed nasotracheal influenza: specific secretory IgA levels and transport of parenterally fed mice. Ann Surg. 2001;233:134 –138.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
25. Kudsk KA. Current aspects of mucosal immunology and its influence by nutrition. Am J Surg.2002; 183:390 –398.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
26. Reese SR, Kudsk KA, Genton L, et al. L-selectin and alpha4beta7 integrin, but not ICAM-1, regulate lymphocyte distribution in gut-associated lymphoid tissue of mice. Surgery.2005; 137:209 –215.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
27. Yamamoto M, Kweon MN, Rennert PD, et al. Role of gut-associated lymphoreticular tissues in antigen-specific intestinal IgA immunity. J Immunol. 2004;173:762 –769.[Abstract/Free Full Text]
28. Ware CF. Network communications: lymphotoxins, LIGHT, and TNF.Annu Rev Immunol. 2005;23:787 –819.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
29. Lo JC, Basak S, James SE, et al. Coordination between NF-B family members p50 and p52 is essential for mediating LTβR signals in the development and organization of secondary lymphoid tissues.Blood. 2006;107:1048 –1055.[Abstract/Free Full Text]
30. Genton L, Kudsk KA, Reese S, Ikeda S. Enteral feeding preserves gut Th-2 cytokines despite MAdCAM-1 blockade. JPEN J Parenter Enteral Nutr. 2005;29:44 –47.[Abstract/Free Full Text]
31. Pascual DW, Stanisz AM, Bost KL. Functional aspects of the peptidergic circuit in mucosal immunity. In: Ogta PL, Lamm ME, McGhee JR, et al, eds. Handbook of Mucosal Immunology. New York, NY: Academic Press Inc; 1994:203 –216.
32. Alverdy J, Stera E, Poticha S, et al. Cholecystokinin modulates mucosal immunoglobulin A function. Surgery.1997; 122:386 –392.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
33. Hanna MK, Zarzaur BL, Fukatsu K, et al. Individual neuropeptides regulate gut-associated lymphoid tissue integrity, intestinal IgA levels, and respiratory antibacterial immunity. JPEN J Parenter Enteral Nutr. 2000;24:261 –269.[Abstract/Free Full Text]
34. Genton L, Kudsk KA. Interactions between the enteric nervous system and mucosal immunity: role of neuropeptides and nutrition. Am J Surg. 2003;186:253 –258.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
35. Zarzaur BL, Wu Y, Fukatsu K, Johnson CD, Kudsk KA. The neuropeptide bombesin improves IgA-mediated mucosal immunity with preservation of gut interleukin-4 in total parenteral nutrition-fed mice. Surgery.2002; 131:59 –65.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
36. DeWitt RC, Wu Y, Renegar KB, King BK, Li J, Kudsk KA. Bombesin recovers gut-associated lymphoid tissue (GALT) and preserves immunity to bacterial pneumonia in TPN-fed mice. Ann Surg.2000; 231:1 –8.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
37. Lorenz RG, Chaplin DD, McDonald KG, McDonough JS, Newberry RD. Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J Immunol.2003; 170:5475 –5482.[Abstract/Free Full Text]
38. Fagarasan S, Muramatsu M, Suzuki K, Nagaoka H, Hiai H, Honjo T. Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora. Science.2002; 298:1424 –1427.[Abstract/Free Full Text]
39. Chin R, Wang J, Fu Y. Lymphoid microenvironment in the gut for immunoglobulin A and inflammation. Immunol Rev.2003; 195:190 –201.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
40. Yamamoto M, Rennert P, McGhee JR, et al. Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract. J Immunol.2000; 164:5184 –5191.[Abstract/Free Full Text]

Discussant

The authors, through their own work and work from others, have shown that LTβR stimulation controls production of IL-4, the adhesion molecule MAdCAM-1, and the control of other key components of GALT. This pathway is important in increasing IgA levels and maintaining mucosal defenses. However, I find one finding in the current paper difficult to understand. The authors showed that blockade of LTβR did not affect IgA levels. The authors state that because the animals were fed, IgA levels continued to remain high; however, I am not sure about this. In particular, the authors made a good case in the background to show how LTβR is important for many components needed for IgA production, and nicely showed a decline in MadCAM-1. In the Discussion section of the paper, the authors begin to address this point, but it may make sense to continue this area of investigation. In particular, did the authors examine or plan to examine the changes in IL-4 or IL-10 with blockade of LTβR? Or did they state plans (in the future) to examine levels of gastrin-releasing peptide (GRP), etc in mice who underwent LTβR blockade?

Additionally, the protein expression of MAdCAM-1 and cytokines was not examined. It may be that the lack of changes in the expression of IgA may be due to the lack of protein changes with PN and that the effects of PN are limited to the RNA level. Do the authors have any additional data regarding this?

Finally, the Peyer's patches have a number of other functional roles aside from IgA production. Did the authors examine any other functional alterations in this model of LTβR blockade?

Author's Response

For the next question, we have demonstrated that the protein expression of MAdCAM-1 is decreased by PN in 2 published experiments. We also have the data that LTβR blockade depresses MAdCAM-1 protein levels in PP. Therefore, it seems unlikely that the lack of changes of IgA levels is due to the lack of protein changes with LTβR-Ig.

For the final question, we did not measure any other functional alterations in this model of LTβR blockade.

Journal of Parenteral and Enteral Nutrition, Vol. 31, No. 5, 358-365 (2007)
DOI: 10.1177/0148607107031005358


CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K. A. Kudsk Jonathan E. Rhoads Lecture: Of Mice and Men... and a Few Hundred Rats JPEN J Parenter Enteral Nutr, July 1, 2008; 32(4): 460 - 473. [Full Text] [PDF]

				K. A. Kudsk, F. E. Gomez, W. Kang, and C. Ueno Enteral Feeding of a Chemically Defined Diet Preserves Pulmonary Immunity but Not Intestinal Immunity: The Role of Lymphotoxin {beta} Receptor JPEN J Parenter Enteral Nutr, November 1, 2007; 31(6): 477 - 481. [Abstract] [Full Text] [PDF]

This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Kang, W. Articles by Maeshima, Y. Search for Related Content PubMed PubMed Citation Articles by Kang, W. Articles by Maeshima, Y. Pubmed/NCBI databases Substance via MeSH Social Bookmarking                   What's this?