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The Role of Dendritic Cells in the Gastrointestinal Field Effect
The Role of Dendritic Cells in the Gastrointestinal Field Effect A. Koscielny, T. Boerner, S. Wehner, C. Kurts, and J.C. Kalff ABSTRACT Introduction. Intestinal manipulation leads to local bowel wall inflammation that subsequently spreads over the entire gastrointestinal tract. Previously, this gastrointestinal field effect had been demonstrated by us in a rodent model. We herein postulated an immunologic mechanism mediated by activated leukocytes. The aim of this study was to investigate the activation, maturation and migration of dendritic cells (DC) of the intestinal smooth muscle following surgical trauma and i.p. lipopolysaccharide challenge. Methods. Mice underwent standardized intestinal manipulation or iP LPS administration and tissues (intestinal muscularis, Peyer’s patches, mesenteric lymph nodes, and spleen) were obtained at various times after manipulation. DC were isolated by tissue digestion and separated by CD11c-iMAG. The harvested DC were analyzed by FACS. The activation pattern of DC was analyzed by polymerase chain reaction. Results. We found a significant increase in DC within the intestinal muscularis, the Peyer’s patches and the mesenteric lymph nodes at 6 and 12 hours following intestinal manipulation and injection of LPS. There was an upregulation of the costimulatory molecules major histocompatibility complex II, CD40, CD80, CD86, and CD205 in the DC after intestinal manipulation. CCR-2, CCR-5, CCR-7, CCL-19, and interleukin-12a were upregulated in a time- and tissue-dependent manner. Conclusion. Intestinal manipulation or LPS challenge induced a recruitment of DC into the muscularis externa and mesenteric lymph nodes combined with an upregulation of costimulatory immunocompetent molecules and migratory surface markers in DCs. These findings demonstrate a precondition for an immunologic response and a possible immunologically mediated gastrointestinal field effect.
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NTESTINAL SURGERY and abdominal trauma result in intestinal dysmotility and ileus, which is known to potentially induce sepsis and multiple organ failure. These functional alterations are accompanied by a time– and trauma-degree– dependent inflammatory cascade within the manipulated jejunal muscularis activating the dense network of resident muscularis macrophages.1 We showed that the sequelae of intestinal surgery may affect distant unmanipulated intestinal segments (colon, stomach) inducing functional changes in gastric emptying and colorectal contractility in vivo and in vitro.2 This phenomenon is called the gastrointestinal field effect. The underlying mechanism has not been elucidated yet. We postulated an immunologic mechanism mediated by activated leukocytes. Interestingly, the intestinal field effect was abolished when the mesenteric lymph was drained off after intestinal manipulation.2 The
aim of this study was to investigate the activation, maturation and migration of dendritic cells (DC) in the intestinal smooth muscle following intestinal manipulation as a precondition for an immunologic response. MATERIALS AND METHODS Male C57BI6-mice weighing 20 to 25 g were obtained from Harlan Laboratories. Two groups of animals were studied: (a) an intestinal manipulation group consisting of animals that had their small From the Department of Surgery (A.K., T.B., S.W., J.C.K.) and Institute of Molecular Medicine and Experimental Immunology (C.K.), University of Bonn Medical School, Bonn, Germany. Address reprint requests to Prof Dr med Joerg C. Kalff, Klinik und Poliklinik für Allgemein-, Viszeral-, Thorax- und Gefässchirurgie, Rheinische Friedrich-Wilhelms-Universität Bonn, Sigmund-FreudStraße 25, D-53125 Bonn, Germany. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.087
Transplantation Proceedings, 38, 1815–1817 (2006)
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Fig 1. Recruitment of dendritic cells to intestinal muscularis layer (IM), Peyer’s patches (PP), mesenteric lymph nodes (MLN) and spleen 6 and 12 hours after intestinal manipulation (man.) compared to the LPS-challenge. (FACS for CD11c-positive cells). bowel subjected to a standardized intestinal manipulation1,2; and (b) a control group of unmanipulated mice. Animals were sacrificed between 0 and 24 hours after manipulation. The intestine was used for isolation and separation of CD11c-positive cells for further experiments, including FACS-analysis for CD11c, CD205, CD40, CD80, CD86, and major histocompatibility complex II, Taqman polymerase chain reaction for CCR2, CCR5, CCR7, interleukin (IL)-12a and CCL-19, T-cell proliferation assays, and ELISA-assay for IL-2 and interferon-␥ (INF-␥). The intestinal tissue was used for in vivo gastrointestinal transit and immunohistochemistry. The isolation and CD11c positive separation of DC from intestinal muscularis (IM) layer, Peyer’s patches, mesenteric lymph nodes (MLN) were performed following previously described protocols including low-density gradient centrifugation and anti-CD11c antibody iMAG selection.3,4 The proliferative responses and chemokine-production by naïve CD8 T-cells from Des. TCR-mice primed by DC isolated from MLN of C57BI6 mice at various times after intestinal manipulation were measured by FACS and ELISA.
RESULTS
We isolated DC from the murine small intestinal muscularis layer at various times after surgical manipulation (1, 3, 6, 12, and 24 hours) and from a control group (Fig 1). After enrichment of CD11c-positive cells, we observed a significantly higher recruitment of DC in the IM and MLN after intestinal manipulation. Immunohistochemistry showed a diffuse infiltration of CD11c-positive cells in the IM at 24 hours compared to control intestine with only few detectable CD11c-positive cells. The control DC expressed low levels of immunologic costimulatory molecules. However, after manipulation the isolated DC showed significantly higher levels of those costimulatory molecules both in IM and MLN (Figs 2 and 3). The DC surface receptors CCR2, CCR5, CCR7, and IL-12 produced by DC also significantly increased after manipulation. Whereas CCR2 and CCR5 decreased 24 hours after manipulation, CCR7 remained high. All these changes in the DC also occurred after LPS challenge, but were less impressive (Fig 4). Proliferative responses of naïve Des. TCR–T-cells on MLN-DC were higher and more effective when DC were
KOSCIELNY, BOERNER, WEHNER ET AL
Fig 2. Expression of costimulatory molecules on DC surface in nonmanipulated mice (ratio of expressing cells to all isolated DC) in FACS. Abbreviations: IM, intestinal muscularis layer; PP, Peyer’s patches; MLN, mesenteric lymph nodes; PMP, peritoneal macrophages.
isolated after manipulation. Additionally, the T cells primed by DC when isolated after manipulation showed increased INF-␥ and IL-2 production. DISCUSSION
This study presented phenotypic and functional data on DC in IM and MLN isolated at various times after intestinal manipulation. We have previously shown that the postoperative inflammatory field effect phenomenon in unmanipulated gastric and colonic muscularis within the rodent is marked by an upregulation of proinflammatory cytokines, adhesion molecules, and inflammatory cells at the traumatized site and also in the unmanipulated stomach and colon.1,2 The precondition for induction of an immunologic response is activation and maturation of DC in peripheral tissues and their migration to local lymph nodes.3– 6 In this study, the DC of IM increased in number and showed upregulated costimulatory immunocompetent molecules and migratory surface markers after intestinal manipulation. Because these isolated DC induced proliferative responses in naïve T cells and influenced their IL-2 and INF-␥ production, the upregulation of costimulatory molecules and chemokine receptors is not only mediated by activation
Fig 3. Expression of costimulatory molecules on DC surface in manipulated mice in FACS (24 hours after intestinal manipulation).
DC IN THE GI FIELD EFFECT
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Fig 4. Intestinal manipulation leads to an upregulation of the chemokine receptors CCR2, CCR5, and CCR7 depending on the posttraumatic time as shown by Taqman PCR.
of DC by local inflammation, but possibly also by antigen presentation. In conclusion, intestinal manipulation or LPS challenge induced recruitment of DC into the intestinal muscularis externa and mesenteric lymph nodes combined with upregulation of costimulatory immunocompetent molecules and migratory surface markers. These findings demonstrated a precondition for an immunologic response and a possible immunologically mediated field effect. REFERENCES 1. Kalff JC, Schraut WH, Simmons RL, et al: Surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus. Ann Surg 228:652, 1998
2. Schwarz NT, Kalff JC, Türler A, et al: Selective jejunal manipulation causes postoperative pan-enteric inflammation and dysmotility. Gastroenterology 126:159, 2004 3. Anjuère F, Luci C, Lebens M, et al: In vivo adjuvant-induced mobilization and maturation of gut dendritic cells after oral administration of cholera toxin. J Immunol 173:5103, 2004 4. Krüger T, Benke D, Eitner F, et al: Identification and functional characterization of dendritic cells in healthy kidney and in experimental glomerulonephritis. J Am Soc Nephrol 15:613, 2004 5. Rimoldi M, Chieppa M, Vulcano M, et al: Intestinal epithelial cells control dendritic cell function. Ann N Y Acad Sci 1029:66, 2004 6. Turnbull EL, Yrlid U, Jenkins CD, et al: Intestinal dendritic cell subsets: differential effects of systemic TLR4 stimualtion on migratory fate and activation in vivo. J Immunol 174:1374, 2005
I
NTESTINAL SURGERY and abdominal trauma result in intestinal dysmotility and ileus, which is known to potentially induce sepsis and multiple organ failure. These functional alterations are accompanied by a time– and trauma-degree– dependent inflammatory cascade within the manipulated jejunal muscularis activating the dense network of resident muscularis macrophages.1 We showed that the sequelae of intestinal surgery may affect distant unmanipulated intestinal segments (colon, stomach) inducing functional changes in gastric emptying and colorectal contractility in vivo and in vitro.2 This phenomenon is called the gastrointestinal field effect. The underlying mechanism has not been elucidated yet. We postulated an immunologic mechanism mediated by activated leukocytes. Interestingly, the intestinal field effect was abolished when the mesenteric lymph was drained off after intestinal manipulation.2 The
aim of this study was to investigate the activation, maturation and migration of dendritic cells (DC) in the intestinal smooth muscle following intestinal manipulation as a precondition for an immunologic response. MATERIALS AND METHODS Male C57BI6-mice weighing 20 to 25 g were obtained from Harlan Laboratories. Two groups of animals were studied: (a) an intestinal manipulation group consisting of animals that had their small From the Department of Surgery (A.K., T.B., S.W., J.C.K.) and Institute of Molecular Medicine and Experimental Immunology (C.K.), University of Bonn Medical School, Bonn, Germany. Address reprint requests to Prof Dr med Joerg C. Kalff, Klinik und Poliklinik für Allgemein-, Viszeral-, Thorax- und Gefässchirurgie, Rheinische Friedrich-Wilhelms-Universität Bonn, Sigmund-FreudStraße 25, D-53125 Bonn, Germany. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.06.087
Transplantation Proceedings, 38, 1815–1817 (2006)
1815
1816
Fig 1. Recruitment of dendritic cells to intestinal muscularis layer (IM), Peyer’s patches (PP), mesenteric lymph nodes (MLN) and spleen 6 and 12 hours after intestinal manipulation (man.) compared to the LPS-challenge. (FACS for CD11c-positive cells). bowel subjected to a standardized intestinal manipulation1,2; and (b) a control group of unmanipulated mice. Animals were sacrificed between 0 and 24 hours after manipulation. The intestine was used for isolation and separation of CD11c-positive cells for further experiments, including FACS-analysis for CD11c, CD205, CD40, CD80, CD86, and major histocompatibility complex II, Taqman polymerase chain reaction for CCR2, CCR5, CCR7, interleukin (IL)-12a and CCL-19, T-cell proliferation assays, and ELISA-assay for IL-2 and interferon-␥ (INF-␥). The intestinal tissue was used for in vivo gastrointestinal transit and immunohistochemistry. The isolation and CD11c positive separation of DC from intestinal muscularis (IM) layer, Peyer’s patches, mesenteric lymph nodes (MLN) were performed following previously described protocols including low-density gradient centrifugation and anti-CD11c antibody iMAG selection.3,4 The proliferative responses and chemokine-production by naïve CD8 T-cells from Des. TCR-mice primed by DC isolated from MLN of C57BI6 mice at various times after intestinal manipulation were measured by FACS and ELISA.
RESULTS
We isolated DC from the murine small intestinal muscularis layer at various times after surgical manipulation (1, 3, 6, 12, and 24 hours) and from a control group (Fig 1). After enrichment of CD11c-positive cells, we observed a significantly higher recruitment of DC in the IM and MLN after intestinal manipulation. Immunohistochemistry showed a diffuse infiltration of CD11c-positive cells in the IM at 24 hours compared to control intestine with only few detectable CD11c-positive cells. The control DC expressed low levels of immunologic costimulatory molecules. However, after manipulation the isolated DC showed significantly higher levels of those costimulatory molecules both in IM and MLN (Figs 2 and 3). The DC surface receptors CCR2, CCR5, CCR7, and IL-12 produced by DC also significantly increased after manipulation. Whereas CCR2 and CCR5 decreased 24 hours after manipulation, CCR7 remained high. All these changes in the DC also occurred after LPS challenge, but were less impressive (Fig 4). Proliferative responses of naïve Des. TCR–T-cells on MLN-DC were higher and more effective when DC were
KOSCIELNY, BOERNER, WEHNER ET AL
Fig 2. Expression of costimulatory molecules on DC surface in nonmanipulated mice (ratio of expressing cells to all isolated DC) in FACS. Abbreviations: IM, intestinal muscularis layer; PP, Peyer’s patches; MLN, mesenteric lymph nodes; PMP, peritoneal macrophages.
isolated after manipulation. Additionally, the T cells primed by DC when isolated after manipulation showed increased INF-␥ and IL-2 production. DISCUSSION
This study presented phenotypic and functional data on DC in IM and MLN isolated at various times after intestinal manipulation. We have previously shown that the postoperative inflammatory field effect phenomenon in unmanipulated gastric and colonic muscularis within the rodent is marked by an upregulation of proinflammatory cytokines, adhesion molecules, and inflammatory cells at the traumatized site and also in the unmanipulated stomach and colon.1,2 The precondition for induction of an immunologic response is activation and maturation of DC in peripheral tissues and their migration to local lymph nodes.3– 6 In this study, the DC of IM increased in number and showed upregulated costimulatory immunocompetent molecules and migratory surface markers after intestinal manipulation. Because these isolated DC induced proliferative responses in naïve T cells and influenced their IL-2 and INF-␥ production, the upregulation of costimulatory molecules and chemokine receptors is not only mediated by activation
Fig 3. Expression of costimulatory molecules on DC surface in manipulated mice in FACS (24 hours after intestinal manipulation).
DC IN THE GI FIELD EFFECT
1817
Fig 4. Intestinal manipulation leads to an upregulation of the chemokine receptors CCR2, CCR5, and CCR7 depending on the posttraumatic time as shown by Taqman PCR.
of DC by local inflammation, but possibly also by antigen presentation. In conclusion, intestinal manipulation or LPS challenge induced recruitment of DC into the intestinal muscularis externa and mesenteric lymph nodes combined with upregulation of costimulatory immunocompetent molecules and migratory surface markers. These findings demonstrated a precondition for an immunologic response and a possible immunologically mediated field effect. REFERENCES 1. Kalff JC, Schraut WH, Simmons RL, et al: Surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus. Ann Surg 228:652, 1998
2. Schwarz NT, Kalff JC, Türler A, et al: Selective jejunal manipulation causes postoperative pan-enteric inflammation and dysmotility. Gastroenterology 126:159, 2004 3. Anjuère F, Luci C, Lebens M, et al: In vivo adjuvant-induced mobilization and maturation of gut dendritic cells after oral administration of cholera toxin. J Immunol 173:5103, 2004 4. Krüger T, Benke D, Eitner F, et al: Identification and functional characterization of dendritic cells in healthy kidney and in experimental glomerulonephritis. J Am Soc Nephrol 15:613, 2004 5. Rimoldi M, Chieppa M, Vulcano M, et al: Intestinal epithelial cells control dendritic cell function. Ann N Y Acad Sci 1029:66, 2004 6. Turnbull EL, Yrlid U, Jenkins CD, et al: Intestinal dendritic cell subsets: differential effects of systemic TLR4 stimualtion on migratory fate and activation in vivo. J Immunol 174:1374, 2005