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Interactions between the peripheral nervous system and resident macrophages (MMs) modulate intestinal homeostatic functions. Activation of β2-adrenergic receptors on MMs has been shown to reduce bacterial challenges. These MMs are also crucial for the development of bowel inflammation in postoperative ileus (POI), an iatrogenic, noninfectious inflammation-based motility disorder. However, the role of the sympathetic nervous system (SNS) in the immune modulation of these MMs during POI or other noninfectious diseases is largely unknown. By employing 6-OHDA-induced denervation, we investigated the changes in the muscularis externa by RNA-seq, quantitative PCR, and flow cytometry. Further, we performed transcriptional phenotyping of sorted CX3CR1+ MMs and ex vivo LPS/M-CSF stimulation on these MMs. By combining denervation with a mouse POI model, we explored distinct changes on CX3CR1+ MMs as well as in the muscularis externa and their functional outcome during POI. Our results identify SNS as an important mediator in noninfectious postoperative inflammation. Upon denervation, MMs anti-inflammatory genes were reduced, and the muscularis externa profile is shaped toward a proinflammatory status. Further, denervation reduced MMs anti-inflammatory genes also in the early phase of POI. Finally, reduced leukocyte infiltration into the muscularis led to a quicker recovery of bowel motility in the late phase of POI.

Postoperative ileus (POI) is triggered by an innate immune response in the muscularis externa (ME) and is accompanied by bacterial translocation. Bacteria can trigger an innate immune response via toll-like receptor (TLR) activation, but the latter’s contribution to POI has been disproved for several TLRs, including TLR2 and TLR4. Herein we investigated the role of double-stranded RNA detection via TLR3 and TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling pathway in POI. POI was induced by small bowel intestinal manipulation in wt, TRIF-/-, TLR3-/-, type I interferon receptor-/- and interferon-β reporter mice, all on C57BL/6 background, and POI severity was quantified by gene expression analysis, gastrointestinal transit and leukocyte extravasation into the ME. TRIF/TLR3 deficiency reduced postoperative ME inflammation and prevented POI. With bone marrow transplantation, RNA-sequencing, flow cytometry and immunohistochemistry we revealed a distinct TLR3-expressing radio-resistant MHCIIhiCX3CR1- IBA-1+ resident macrophage population within the deep myenteric plexus. TLR3 deficiency in these cells, but not in MHCIIhiCX3CR1+ macrophages, reduced cytokine expression in POI. While this might not be an exclusive macrophage-privileged pathway, the TLR3/TRIF axis contributes to proinflammatory cytokine production in MHCIIhiCX3CR1- IBA-1+ macrophages during POI. Deficiency in TLR3/TRIF protects mice from POI. These data suggest that TLR3 antagonism may prevent POI in humans.

Postoperative ileus (POI) is an intestinal dysmotility frequently occurring after abdominal surgery. An orchestrated neuroimmune response within the muscularis externa (ME) involves activation of resident macrophages, enteric glia and infiltration of blood-derived leukocytes. Interleukin-1 receptor type-I (IL1R1) signalling on enteric glia has been shown to be involved in POI development. Herein we investigated the distinct role of the IL1R1 ligands interleukin (IL) -1α and IL-1β and focused on the mechanism of IL-1β production. IL-1α and IL-1β deficient mice were protected from POI. Bone-marrow transplantation studies indicated that IL-1α originated from radio-resistant cells while IL-1β was released from the radio-sensitive infiltrating leukocytes. Mouse strains deficient in inflammasome formation identified the absent in melanoma 2 (AIM2) inflammasome to be crucial for IL-1β production in POI. Mechanistically, antibiotic-treated mice revealed a prominent role of the microbiome in IL-1β production. Our study provides new insights into distinct roles of IL-1α and IL-1β signalling during POI. While IL-1α release is most likely an immediate passive response to the surgical trauma, IL-1β production depends on AIM2 inflammasome formation and the microbiome. Selective interaction in this pathway might be a promising target to prevent POI in surgical patients.

Postoperative ileus (POI) is an intestinal dysmotility frequently occurring after abdominal surgery. An orchestrated neuroimmune response within the muscularis externa (ME) involves activation of resident macrophages, enteric glia and infiltration of blood-derived leukocytes. Interleukin-1 receptor type-I (IL1R1) signalling on enteric glia has been shown to be involved in POI development. Herein we investigated the distinct role of the IL1R1 ligands interleukin (IL) -1α and IL-1β and focused on the mechanism of IL-1β production. IL-1α and IL-1β deficient mice were protected from POI. Bone-marrow transplantation studies indicated that IL-1α originated from radio-resistant cells while IL-1β was released from the radio-sensitive infiltrating leukocytes. Mouse strains deficient in inflammasome formation identified the absent in melanoma 2 (AIM2) inflammasome to be crucial for IL-1β production in POI. Mechanistically, antibiotic-treated mice revealed a prominent role of the microbiome in IL-1β production. Our study provides new insights into distinct roles of IL-1α and IL-1β signalling during POI. While IL-1α release is most likely an immediate passive response to the surgical trauma, IL-1β production depends on AIM2 inflammasome formation and the microbiome. Selective interaction in this pathway might be a promising target to prevent POI in surgical patients.

Postoperative ileus (POI) is a common complication after abdominal surgery characterized by motility disturbances leading to increased morbidity and mortality in surgical patients. Intestinal manipulation of the murine small bowel is an established animal model resulting in an increased postsurgical inflammation within the intestinal muscular externa and a delayed gastrointestinal transit. Some analgesics have been shown to affect inflammation. In this study, we compared the immunomodulatory effects of two different analgesics. Mice were treated with tramadol, metamizole or saline as a control in our established POI model. The postoperative inflammatory response was assessed by gene expression of pro-inflammatory cytokines at different time points and immunocytes extravasation into the muscularis externa. Functional motility analyses were performed by a gastrointestinal transit measurement. Metamizole application reduced the pro-inflammatory response after surgery and improved gastrointestinal motility, while tramadol showed no alteration in cytokine gene expression, influx of immunocytes and gastrointestinal transit compared with the controls. In conclusion. we suggest tramadol as analgesia in immunological studies on POI in mice as it does not affect the underlying inflammation of POI.

Objective: Postoperative ileus (POI) is an inflammation-mediated complication of abdominal surgery, characterized by intestinal dysmotility and leukocyte infiltration into the muscularis externa (ME). Previous studies indicated that interleukin (IL)-10 is crucial for the resolution of a variety of inflammation-driven diseases. Herein, we investigated how IL-10 affects the postoperative ME inflammation and found an unforeseen role of IL-10 in POI. Design: POI was induced by a standardized intestinal manipulation (IM) in C57BL/6 and multiple transgenic mouse strain including C-C motif chemokine receptor 2-/-, IL-10-/-, and LysMcre/IL-10fl/fl mice. Leukocyte infiltration, gene and protein expression of cytokines, chemokines, and macrophage differentiation markers as well as intestinal motility were analyzed. IL-10 serum levels in surgical patients were determined by ELISA. Results: IL-10 serum levels were increased in patient after abdominal surgery. In mice, a complete or leucocyte-restricted IL-10 deficiency ameliorated POI and reduced the postoperative ME neutrophil infiltration. Infiltrating monocytes were identified as main IL-10 producers and undergo IL-10-dependent M2 polarization. Interestingly, M2 polarization is not crucial to POI development as abrogation of monocyte infiltration did not prevent POI due to a compensation of the IL-10 loss by resident macrophages and neutrophils. Organ culture studies demonstrated that IL-10 deficiency impeded neutrophil migration toward the surgically traumatized ME. This mechanism is mediated by reduction of neutrophil attracting chemokines. Conclusion: Monocyte-derived macrophages are the major IL-10 source during POI. An IL-10 deficiency decreases the postoperative expression of neutrophil-recruiting chemokines, consequently reduces the neutrophil extravasation into the postsurgical bowel wall, and finally protects mice from POI.

Aim To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus. Methods C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay. Results Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI. Conclusion Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery.

Mechanical trauma of the gut is an unavoidable event in abdominal surgery. Former studies demonstrated that intestinal manipulation induces a strong inflammation within the tunica muscularis. We hypothesized that mechanical strain initiates or aggravates proinflammatory responses in intestinal smooth muscle cells (iSMC) or macrophages. First, an appropriate isolation and culture method for neonatal rat iSMC was established. Purified iSMC and primary peritoneal macrophages (pMacs) were subjected to static or cyclic strain, and gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-6, and IL-1β was analyzed by quantitative PCR. Supernatants from stretched iSMC were transferred to untreated pMacs or contrariwise, and medium transfer-triggered inflammatory gene expression was measured in unstretched cells. Finally, we investigated the synergistic effect of static strain on LPS-induced proinflammatory gene expression. Although cyclic strain failed, static strain significantly induced iNOS, COX-2, and IL-1β mRNA in iSMC. pMacs showed an increase in all inflammatory genes investigated as well as macrophage inflammatory protein (MIP)-1α and MIP-2 mRNA after static strain. Both cell entities liberated unknown mediators in response to stretch that mutually stimulated iNOS gene expression. Finally, mechanostimulation amplified LPS-induced iNOS and IL-1β gene expression in iSMC as well as COX-2 and IL-6 mRNA in pMacs. In conclusion, static strain initiates proinflammatory gene expression in iSMC and pMacs and triggers a bidirectional paracrine communication between both cultured cell entities via the liberation of unknown mediators. Furthermore, static strain synergistically operates with Toll-like receptor 4 ligation in a cell-specific manner. Hence, this study demonstrates that mechanical strain functions as an immunomodulatory stimulus in abdominal cells.

An in vivo model was used to investigate the role of cathepsins in mouse intestine after mechanical manipulation. Inspection of different intestine segments by immunofluorescence microscopy provided evidence for a local release of cathepsin B from cells of individual gut sections shortly after traumatic injury. Densitometry of immunoblots ruled out alterations in cathepsin B expression levels. Because similar results were obtained with both mouse and rat intestine trauma models, we were interested in identifying potential targets of released cathepsin B in early post-traumatic phases. Immunoblotting revealed initial declines followed by an increase in protein levels of claudin-1 and E-cadherin, indicating that tight junctions and cell-cell adhesions were only transiently compromised by surgical trauma. Apical aminopeptidase N and dipeptidyl peptidase IV were only slightly affected, whereas basolateral low-density lipoprotein receptors were strongly up-regulated in response to trauma. As potential targets of cathepsin B released from injured cells, we identified collagen IV and laminin of the basement membrane that was damaged during initial post-traumatic stages. Because increased collagen IV expression was observed in the intestine of cathepsin B-deficient animals, we propose a direct role of cathepsin B in that it contributes to acute post-traumatic extracellular matrix damage and may thereby facilitate onset of post-operative ileus.

Resolution of inflammation is an active counter-regulatory mechanism involving polyunsaturated fatty acid-derived proresolving lipid mediators. Postoperative intestinal motility disturbances, clinically known as postoperative ileus, occur frequently after abdominal surgery and are mediated by a complex inflammation of the intestinal muscularis externa. Herein, we tested the hypothesis that proresolving lipid mediators are involved in the resolution of postoperative ileus. In a standardized experimental model of postoperative ileus, we detected strong expression of 12/15-lipoxygenase within the postoperative muscularis externa of C57BL/6 mice, predominately located within CX3CR1+/Ly6C+ infiltrating monocytes rather than Ly6G+ neutrophils. Mass spectrometry analyses demonstrated that a 12/15-lipoxygenase increase was accompanied by production of docosahexaenoic acid-derived lipid mediators, particularly protectin DX and resolvin D2, and their common precursor 17-hydroxy docosahexaenoic acid. Perioperative administration of protectin DX, but not resolvin D2 diminished blood-derived leukocyte infiltration into the surgically manipulated muscularis externa and improved the gastrointestinal motility. Flow cytometry analyses showed impaired Ly6G+/Ly6C+ neutrophil extravasation after protectin DX treatment, whereas Ly6G-/Ly6C+ monocyte numbers were not affected. 12/15-lipoxygenase-deficient mice, lacking endogenous protectin DX synthesis, demonstrated increased postoperative leukocyte levels. Preoperative intravenous administration of a docosahexaenoic acid-rich lipid emulsion reduced postoperative leukocyte infiltration in wild-type mice but failed in 12/15-lipoxygenase-deficient mice mice. Protectin DX application reduced leukocyte influx and rescued 12/15-lipoxygenase-deficient mice mice from postoperative ileus. In conclusion, our results show that 12/15-lipoxygenase mediates postoperative ileus resolution via production of proresolving docosahexaenoic acid-derived protectin DX. Perioperative, parenteral protectin DX or docosahexaenoic acid supplementation, as well as modulation of the 12/15-lipoxygenase pathway, may be instrumental in prevention of postoperative ileus.

Background Abdominal surgery results in neuronal mediator release and subsequent acute intestinal hypomotility. This phase is followed by a longer lasting inflammatory phase resulting in postoperative ileus (POI). Calcitonin gene-related peptide (CGRP) has been shown to induce motility disturbances and in addition may be a candidate mediator to elicit neurogenic inflammation. We hypothesized that CGRP contributes to intestinal inflammation and POI. Methods The effect of CGRP in POI was tested in mice treated with the highly specific CGRP receptor antagonist BIBN4096BS and in CGRP receptor-deficient (RAMP-1−/−) mice. POI severity was analyzed by cytokine expression, muscular inflammation and gastrointestinal (GI) transit. Peritoneal and muscularis macrophages and mast cells were analyzed for CGRP receptor expression and functional response to CGRP stimulation. Key Results Intestinal manipulation (IM) resulted in CGRP release from myenteric nerves, and a concurrent increased interleukin (IL)-6 and IL-1β transcription and leukocyte infiltration in the muscularis externa and increased GI transit time. CGRP potentiates IM-induced cytokine transcription within the muscularis externa and peritoneal macrophages. BIBN4096BS reduced cytokine levels and leukocyte infiltration and normalized GI transit. RAMP1−/− mice showed a significantly reduced leukocyte influx. CGRP receptor was expressed in muscularis and peritoneal macrophages but not mast cells. CGRP mediated macrophage activation but failed to induce mast cell degranulation and cytokine expression. Conclusions & Inferences CGRP is immediately released during abdominal surgery and induces a neurogenic inflammation via activation of abdominal macrophages. BIBN4096BS prevented IM-induced inflammation and restored GI motility. These findings suggest that CGRP receptor antagonism could be instrumental in the prevention of POI.

Background & Aims Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. Methods We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2 −/− , TLR-4 −/− , TLR-2/4 −/− , MyD88 −/− , MyD88/TLR adaptor molecule 1 −/− , interleukin-1 receptor [IL-1R1] −/− , and interleukin (IL)-18 −/− mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. Results Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1 −/− mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1β before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1β were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1β stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. Conclusions IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI.

Postoperative ileus (POI) is an iatrogenic complication of abdominal surgery, mediated by a severe inflammation of the muscularis externa (ME). Previously, we demonstrated that intravenous application of the tetravalent guanylhydrazone semapimod (CNI-1493) prevents POI, but the underlying mode of action could not definitively be confirmed. Herein, we investigated the effect of a novel orally active salt of semapimod (CPSI-2364) on POI in rodents and distinguished between its inhibitory peripheral and stimulatory central nervous effects on anti-inflammatory vagus nerve signaling. Distribution of radiolabeled orally administered CPSI-2364 was analyzed by whole body autoradiography and liquid scintillation counting. POI was induced by intestinal manipulation with or without preoperative vagotomy. CPSI-2364 was administered preoperatively via gavage in a dose- and time-dependent manner. ME specimens were assessed for p38-MAP kinase activity by immunoblotting, neutrophil extravasation, and nitric oxide production. Furthermore, in vivo gastrointestinal (GIT) and colonic transit were measured. Autoradiography demonstrated a near-exclusive detection of CPSI-2364 within the gastrointestinal wall and contents. Preoperative CPSI-2364 application significantly reduced postoperative neutrophil counts, nitric oxide release, GIT deceleration, and delay of colonic transit time, while intraoperatively administered CPSI-2364 failed to improve POI. CPSI-2364 also prevents postoperative neutrophil increase and GIT deceleration in vagotomized mice. Orally administered CPSI-2364 shows a near-exclusive dispersal in the gastrointestinal tract and effectively reduces POI independently of central vagus nerve stimulation. Its efficacy after single oral dosage affirms CPSI-2364 treatment as a promising strategy for prophylaxis of POI.

Introduction: Inflammatory molecular and cellular events within the postsurgical intestinal muscularis cause postoperative ileus (POI). Translocating bacteria can lead to microbial invasion, escalation of immune defense and subsequently to conspicuous gastrointestinal dysfunction. Our goal was to determine[for full text, please go to the a.m. URL], 128. Kongress der Deutschen Gesellschaft für Chirurgie

Introduction: Abdominal surgery results in the inflammation of the tunica muscularis (ME), subsequently leading to postoperative ileus (POI) [1]. Previously, we demonstrated that inhibition of macrophage function and cytokine production prevents rodents from POI [2]. Omega-3 and omega-6 polyunsaturated fatty acids (PUFA) are known to exert anti- or proinflammatory effects, respectively. The aim of this study was to analyze the effect of omega-3 and omega-6 PUFA on POI in rodents. Methods: Male rats and mice underwent intestinal manipulation (IM) after 5 days preoperative enteral or parenteral application of vehicle, omega-3 (Omegaven®) or omega-6 (Lipovenos®-MCT) PUFA-enriched emulsions (1 ml/100 g bodyweight, once daily). Erythrocyte membranes were analyzed by gas chromatography for fatty acid composition. Inflammatory activity was determined by detection of neutrophils 24 h after IM. Production of nitric oxide (NO) from ME specimen was determined by Griess reaction. Contractility of jejunal ME strips was measured in vitro in an organ bath setup. Gastrointestinal and colonic transits were determined in vivo. Results: Omegaven treatment significantly increased omega-3 to omega-6 PUFA rates in erythrocytes (1 : 9), compared to untreated controls (1 : 15). Furthermore, Omegaven reduced neutrophil levels (–35 %) and NO production (–38 %) after IM and improved contractility and gastrointestinal and colonic transit time. Interestingly, enteral Omegaven treatment showed increased NO production and tended toward increased neutrophils levels. In all experiments, Lipovenos-MCT treatment solely showed a trend to reduced inflammation and improved motility. Conclusion: Perioperative parenteral omega-3 PUFA enriched nutrition reduces intestinal inflammation and prevents postoperative ileus.

Einleitung: Das mechanische Darmtrauma ist eine unausweichliche Konsequenz chirurgischer Eingriffe und führt bei abdominellen Operationen über eine massive Entzündung der Darmwand zum postoperativen Ileus. Als Auslöser werden sowohl das mechanische Trauma als auch die Translokation[for full text, please go to the a.m. URL], 126. Kongress der Deutschen Gesellschaft für Chirurgie

Introduction: The mechanical trauma is an iatrogenic consequence of abdominal surgery that results in a massive inflammation of the entire bowel wall, subsequently leading to postoperative ileus 1, 2. In addition to the trauma, bacterial translocation and LPS challenge are discussed as initiator of this inflammation. Although several cell types are involved, resident muscularis macrophages and smooth muscle cells play a central role. The aim of this study was to investigate the cellular and molecular reactions of both cell types after LPS-challenge or mechanical trauma. Methods: Rat intestinal (iSMC) and vascular (vSMC) smooth muscle cells and peritoneal macrophages (pMacs) were repetitively stretched for 6 hours in the presence or absence of 100 ng/ml LPS. Gene expression of iNOS, COX-2 and interleukin-6 was analyzed by quantitative PCR. In another experimental setting, intercellular inflammatory stimulation was analyzed after transfer of conditioned culture media. Therefore, media from stretched cells were transferred to unstretched cells — from SMC to macrophages or vice versaand MIP-1a, MIP-2, COX-2, IL-6 and iNOS expression was analyzed after 6 hours. Results: Mechanical stretch does not alter iNOS, COX-2 or IL-6 gene expression in iSMC compared to unstretched controls. However, LPS-challenge induced a significant gene expression of IL-6 (3-fold) and iNOS (292-fold) in iSMC. LPS and stretch together led to and further significant increase of IL-6 (5-fold) and iNOS (1250-fold). However, IL-6 expression was not altered in vSMC cultures by stretch or LPS stimulation, whereas COX-2 expression was increased by stretch (7.8-fold) and enhanced by stretch with LPS (15.4-fold). Stretch together with LPS stimulation did not increase the LPS induced iNOS expression (141-fold) in vSMC. In pMacs, gene expression was not altered by mechanical stretch but all genes were strongly increased after LPS induction. LPS induced proinflammatory gene expression was significantly enhanced in combination with mechanical stretch. The transfer of conditioned media from stretched iSMC induced a significant increase in MIP-2, iNOS and COX-2 expression in pMacs. On the other way, no effect on gene expression was observed in iSMC after transfer of media from stretched pMacs. Conclusion: A mechanical trauma does not induce a proinflammatory gene expression in macrophages and iSMC but it enhances LPS induced strong inflammatory reactions in both cell types. However, in response to a mechanical trauma, iSMC liberate unknown mediators that initiate a proinflammatory reaction in pMacs. Interestingly, vascular and intestinal smooth muscle cells differ in their molecular responses to stretch. Finally, LPS stimulation and a mechanical trauma together induce a massive inflammatory reaction in intestinal smooth cells and macrophages. The investigation of the molecular basics could help to identify targets responsible for the overwhelming inflammation in postoperative inflammations.

Background: Abdominal surgery results in a molecular and cellular inflammatory response in the intestine, leading to postoperative ileus. It was hypothesised that resident macrophages within the intestinal muscularis have an important role in this local inflammation. Aims: To investigate whether chemical or genetic depletion of resident muscularis macrophages would lead to a reduction in the local inflammation and smooth-muscle dysfunction. Methods: Two rodent models were used to deplete and inactivate macrophages: (1) a rat model in which resident macrophages were depleted by chlodronate liposomes; (2) a model of mice with osteopetrosis mice, completely lacking the resident muscularis macrophages, used as an additional genetic approach. Animals with normal or altered intestinal macrophages underwent surgical intestinal manipulation. The inflammatory response was investigated by quantitative reverse transcriptase-polymerase chain reaction for mRNA of MIP-1α, interleukin (IL)1β, IL6, intracellular adhesion molecule 1 (ICAM-1) and monocyte chemotractant protein 1 (MCP)-1 in the isolated small bowel muscularis. In addition, muscularis whole mounts were used for histochemical and immunohistochemical analysis to quantify leucocyte infiltration and detect cytokine expression. Subsequently, in vitro muscle contractility and in vivo gastrointestinal transit were measured. Results: Both models resulted in markedly decreased expression of MIP-1α, IL1β, IL6, ICAM-1 and MCP-1 after manipulation compared with controls. In addition to this decrease in inflammatory mediators, recruitment of leucocytes into the muscularis was also diminished. Macrophage-altered animals had near normal in vitro jejunal circular muscle function and gastrointestinal transit despite surgical manipulation. Conclusions: Resident intestinal muscularis macrophages are initially involved in inflammatory responses resulting in postoperative ileus. Depletion and inactivation of the muscularis macrophage network prevents postoperative ileus.

Background and aims: Abdominal surgery results in a massive inflammatory reaction within the intestinal wall leading to postoperative ileus. Results of previous investigations suggested that resident macrophages within the intestinal muscularis play an important role in the local inflammatory process. Aim of this study was to determine if intestinal manipulation (IM) of the homozygous op/op−/− mouse compared to normal C57BL6-mice respectively heterozygous op/op+/− mice would lead to a reduction of the local inflammation and subsequently to normalization of smooth muscle function. Very important in this case is the complete absence of macrophages in the intestinal tunica muscularis of the op/op−/− mouse, having only a reduced number of macrophages in tunica mucosa and other organs. Methods: Three groups of mice were studied: homozygous op/op−/− mice, C57BL6-mice and heterozygous op/op+/− mice (n = 5/group) underwent a standardized model of intestinal manipulation and were examined on the 1 postoperative day. Controls were unoperated animals. Jejunal muscularis whole mounts were used for histochemistry to quantify leukocyte populations. Gastrointestinal transit was measured in vivo after ingestion of fluorescently labelled dextrane. Statistical analysis was made by means of student t-test and ANOVA, p < 0.05. Results: Intestinal manipulation led in all three groups to significant increase of phagocytes in the muscularis compared to not manipulated animals. This increase was significantly lower in homozygous osteopetrosis mice (43 times vs. 180 times C57BL6 and 176 times op/op+/- mice). In vivo gastrointestinal transit demonstrated a significant improvement of the known postoperative smooth muscle function inhibition in osteopetrosis mice. Conclusions: These data demonstrate that the local inflammatory response and smooth muscle dysfunction following surgical intestinal trauma are decreased in the osteopetrosis mouse. Therefore, these results point out the role of the resident macrophages in the intestinal muscularis in the pathomechanism of postoperative ileus.

Background and aims: Mechanical trauma is the iatrogenic consequence of abdominal surgical procedures that result in a strong inflammation of the whole intestine and finally in postoperative ileus. Previous investigations of our group demonstrate that the activation of resident intestinal muscularis macrophages is a key event in this local inflammation process. The aim of this study was to determine if intestinal macrophages are directly activated by mechanical trauma and to investigate the underly-ing molecular signalling pathways. Methods: Single cell suspensions were isolated by enzymatic digestion of the muscularis and cultured as pureor co-culture of smooth muscle cells and intestinal macrophages. Cells were analysed for their expression of CD11b and CD163 (macrophages) and α-actin (smooth muscle cells). To receive a sufficient number of macrophages a cell line was gener-ated by retroviral oncogene transfer (v-myc) and characterized by FACS analysis. For the transfer of the (in vivo) surgical trauma, applicated by running compression of the intestine, to cell cultures (in vitro) macrophages were cultured and stretched on silicon membranes (FlexCell® system). Cells were subsequently analysed for their expression of the activation markers MIP-1α and LFA-1 by quantita-tive PCR. Results: In this study we show that primary intestinal muscularis macrophages show a distinct dendritic phenotype in coculture with smooth muscle cells. Immortalisation of these macrophages by retroviral oncogene transfer generates a phagocytic cell line, expressing the macrophage markers F4/80, CD11b and CD14. The imitation of an in vivo surgical trauma by in vitro application of mechanical stretch to intestinal macrophages, leads to a significant upregulation of LFA-1 and MIP-1a expression. Conclusion: We were able-for the first time- to generate an intestinal muscularis macrophage cell line. Furthermore, our results suggest that mechanical stretch directly activates intestinal macrophages, presumably initiating the molecular signalling pathways that lead to postoperative ileus.

The goal was to investigate the neuroimmune axis in irritable bowel syndrome (IBS) by analyzing the neuroendocrine and cellular immune responses to nutrient load.In the fasting state and 20, 40, 70, and 100 min following nutrient load, blood samples were collected and cardiovascular recordings were accomplished in 15 female IBS patients and 15 healthy women. Plasma norepinephrine, prolactin, cortisol, and growth hormone were analyzed, and blood pressure and heart rate responses were measured. The distribution of peripheral leukocytes and lymphocyte subpopulations and the in vitro production of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) after whole blood stimulation with in lipopolysaccharide (LPS) were analyzed.IBS patients demonstrated significantly greater postprandial increases in plasma norepinephrine and systolic blood pressure (p0.05), but no cortisol response. A postprandial redistribution of circulating leukocytes and lymphocyte subpopulations was observed in both groups, including significant increases in the numbers of leukocytes and granulocytes and significant decreases in the numbers of monocytes, T-cells, and natural killer (NK) cells (all p0.05). However, IBS patients demonstrated significantly greater postprandial increases in leukocytes and granulocytes, while changes in the numbers of monocytes and NK cells were significantly diminished (all p0.05). Patients also failed to show the postprandial decrease in the in vitro TNF-alpha production observed in controls. Postprandial norepinephrine concentrations were negatively correlated with NK cell numbers in IBS patients (r= 0.58, p0.05) but not controls.IBS may involve an autonomic hyper-responsiveness to visceral stimuli, which occurs throughout the entire gut, is independent of acutely perceived GI symptoms, and does not necessarily involve HPA axis activation. Women with IBS show altered cellular immune responses to food intake, which may at least in part be mediated by adrenergic mechanisms. Thus, autonomic disturbances may have implications for cellular immune function along the neuroendocrine-immune axis in patients with IBS.

Background and aims: Abdominal surgery results in a massive inflammatory reaction within the intestinal wall leading to postoperative ileus. Results of previous investigations suggested that resident macrophages within the intestinal muscularis play an important role in the local inflammatory process. Aim of this study was to determine if depletion and inactivation of these macrophages would lead to a reduction of the local inflammation and subsequently to a normalization of smooth muscle function. Methods: Sprague-Dawley rats underwent a standardized model of intestinal manipulation. Depletion and inactivation-groups were pretreated i.v. with clodronate-encapsulated-liposomes and gadolinium chloride. Cytokine mRNA expression (IL-Iβ, IL-6, TNF-α) was determined by real-time RT-PCR in isolated bowel wall layers. Muscularis whole mounts were used for histo- and immunohistochemistry to quantify leukocyte populations. Gastrointestinal transit was measured in-vivo after ingestion of fluorescently labelled dextran. In-vitro muscle contractility was measured in a standard organ bath using jejunal muscle strips. Statistical analysis was done with the student t-test and ANOVA, p < 0.05. Results: Pretreatment resulted in 85% depletion of muscularis macrophages. Cytokine mRNA levels showed a maximum increase at 3 hours after manipulation. Pretreament significantly reduced the induction within the muscularis. Intestinal manipulation led to a massive infiltration into the muscularis, which was decreased significantly by pretreatment. The inflammatory reaction was followed by a significant decline in muscle contractility in untreated animals, but not significantly altered after pretreament. This functional improvement could also be demonstrated by analysis of gastrointestinal transit. Conclusions: These data show that depletion and inactivation of resident muscularis macrophages decrease the local inflammatory response and smooth muscle dysfunction following surgical intestinal trauma. Therefore, this study shows for the first time that resident macrophages of the intestinal muscularis are causally involved in the local inflammatory reaction and subsequent functional impairment of the smooth muscle, which leads to postoperative ileus.

Introduction: Abdominal surgery results in a massive inflammation within the intestinal wall leading to postoperative ileus. Previous results suggested that resident macrophages within the intestinal muscularis play an important role in the local inflammatory process. Aim of this study was to determine if depletion and inactivation of these macrophages would lead to a reduction of the inflammation and subsequently to normalization of smooth muscle function. Methods: Rats underwent intestinal manipulation. Depletion and inactivation-groups were pretreated i.v. with clodronate-encapsulated-liposomes and gadolinium chloride. Cytokine mRNA expression (IL-1b, IL-6, TNF-a) was determined by real-time RT-PCR in isolated bowel wall layers. Muscularis whole mounts were used for histo- and immunohistochemistry to quantify leukocytes. In-vivo gastrointestinal transit and in-vitro muscle contractility in a standard organ bath were measured. Statistical analysis was done with the student t-test and ANOVA, p Results: Pretreatment resulted in 85% depletion of muscularis macrophages. Intestinal manipulation led to a massive leukocyte recruitment (neutrophils (PMN): 155.5-fold), a significant up-regulation of cytokines (3h postoperatively: IL-1β: 20.3fold, IL-6: 33.2-fold) and a significant decline in muscle contractility in-vivo and in-vitro. Pretreament significantly reduced the inflammatory reaction (PMN: 75.6%; IL-1β: 63.5%, IL-6: 50.6%) and averted muscle dysfunction. Conclusions: These data show that depletion and inactivation of resident muscularis macrophages decrease the local inflammatory response and smooth muscle dysfunction following surgical intestinal trauma. Therefore, this study shows for the first time that resident macrophages of the intestinal muscularis are causally involved in the local inflammatory reaction and subsequent functional impairment of the smooth muscle, which leads to postoperative ileus.

neuromuscular function by increasing basal tone and response to cholinergic stimulus. These effects are not reversible after short-term observation. It is known that temperature affects both the ionic conductances and the rate constants modulating the action potential propagation in giant squid axon. We hypothesize that temperature gradients, by changing the ion dynamics, could influence the electrochemical behaviour of intestinal muscle, with a memory effect which should require long time to be deleted. Further studies are needed to establish the exact involved mechanisms in order to better understand clinical consequences of hypothermia during abdominal surgery.