Andreas Türler

Intra-Abdominal Activation of a Local Inflammatory Response Within the Human Muscularis Externa During Laparotomy

ObjectiveTo investigate the initiation of a complex inflammatory response within the human intestinal muscularis intraoperatively so as to determine the clinical applicability of the inflammatory hypothesis of postoperative ileus. Summary Background DataMild intestinal manipulation in rodents initiates the activation of transcription factors, upregulates proinflammatory cytokines, and increases the release of kinetically active mediators (nitric oxide and prostaglandins), all of which results in the recruitment of leukocytes and a suppression in motility (i.e., postoperative ileus). MethodsHuman small bowel specimens were harvested during abdominal procedures at various times after laparotomy. Histochemical and immunohistochemical techniques were applied to intestinal muscularis whole-mounts. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed for interleukin (IL)-6, IL-1&bgr;, tumor necrosis factor (TNF)-&agr;, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Signal transducers and activators of transcription (STAT) protein phosphorylation was determined by electromobility shift assay. Organ bath experiments were performed on jejunal circular smooth muscle strips. GW274150C and DFU were used in vitro as iNOS and COX-2 inhibitors. ResultsNormal human muscularis externa contained numerous macrophages that expressed increased lymphocyte function associated antigen-1 (LFA-1) immunoreactivity as a function of intraoperative time. RT-PCR demonstrated a time-dependent induction of IL-6, IL-1&bgr;, TNF-&agr;, iNOS, and COX-2 mRNAs within muscularis extracts after incision. Mediators were localized to macrophages with STAT protein activation in protein extracts demonstrating local IL-6 functional activity. DFU alone or in combination with GW274150C increased circular muscle contractility. Specimens harvested after reoperation developed leukocytic infiltrates and displayed diminished in vitro muscle contractility. ConclusionsThese human data demonstrate that surgical trauma is followed by resident muscularis macrophage activation and the upregulation, release, and functional activity of proinflammatory cytokines and kinetically active mediators.

Colonic Postoperative Inflammatory Ileus in the Rat

ObjectiveTo investigate local inflammatory events within the colonic muscularis as a causative factor of postoperative ileus. Summary Background DataSurgically induced intestinal muscularis inflammation has been hypothesized as a mechanism for postoperative ileus. The colon is a crucial component for recovery of gastrointestinal motor function after surgery but remains unaddressed. The authors hypothesize that colonic manipulation initiates inflammatory events that directly mediate postoperative smooth muscle dysfunction. MethodsRats underwent colonic manipulation. In vivo transit and colonic motility was estimated using geometric center analysis and intraluminal pressure monitoring. Leukocyte extravasation was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase–polymerase chain reaction. In vitro circular muscle contractility was assessed in a standard organ bath. The relevance of iNOS and COX-2 inhibition was determined using DFU or L-NIL perfusion. ResultsColonic manipulation resulted in a massive leukocyte recruitment and an increase in inflammatory mRNA expression. This inflammatory response was associated with an impairment of in vivo motor function and an inhibition of in vitro smooth muscle contractility (56%). L-NIL but not DFU significantly ameliorated smooth muscle dysfunction. ConclusionsThe results provide evidence for a surgically initiated local inflammatory cascade within the colonic muscularis that mediates smooth muscle dysfunction, which contributes to postoperative ileus.

Management of Drug-to-Drug Interactions Between Cyclosporine A and the Protease-Inhibitor Lopinavir/ Ritonavir in Liver-Transplanted HIV-Infected Patients

Highly active antiretroviral therapy (HAART) has improved the life expectancy of HIV‐infected patients, allowing orthotopic liver transplantation as a reasonable treatment option for selected patients with terminal liver disease. Both non‐nucleoside reverse transcriptase inhibitors and protease inhibitors, key elements of HAART, give rise to substantial drug‐to‐drug interactions with immunosuppressive drugs such as tacrolimus and cyclosporine A. After studying 12‐hour pharmacokinetic profiles in 3 HIV‐positive patients after liver transplantation, we describe how dosing of cyclosporine A can be adjusted to maintain effective immunosuppressive drug levels on a daily dosing schedule when ritonavir‐boosted indinavir or lopinavir‐based antiretroviral therapy is given. To avoid toxic drug levels, we used an orally available cyclosporine A formulation prepared from the commercial available intravenous solution, which enabled dose adjustments in 1‐mg increments. Under ritonavir‐boosted HAART, cyclosporine A levels showed markedly altered absorption/elimination characteristics with more or less constant blood‐levels throughout the dosing interval and prolonged elimination half‐lives up to 38 hours. To obtain equivalent areas under the curve of cyclosporine A, daily doses were reduced to 5–20% of the individual standard doses given before initiation of ritonavir‐boosted HAART. Because of the flat absorption/elimination profiles under ritonavir‐boosted HAART cyclosporine A, dosing could be reliably monitored long term by measuring cyclosporine A trough‐levels. (Liver Transpl 2004;10:939–944.)

Leukocyte-derived inducible nitric oxide synthase mediates murine postoperative ileus.

Objective:To provide evidence that iNOS expression solely in leukocytes plays a role in postoperative ileus. Summary Background Data:Intestinal handling initiates a molecular and cellular muscularis inflammation that has been associated with iNOS expression and ileus. The specific cellular source of iNOS is a matter of speculation. Methods:Chimeric mice were constructed that selectively express the iNOS gene only in their leukocytes or only in their parenchymal cells by lethal radiation and reconstitution with reciprocal bone marrow. Mild intestinal manipulation was used to induce postoperative ileus. Results:Intestinal manipulation caused a significant leukocyte extravasation into the muscularis of all groups. Postoperative iNOS mRNA expression was evident in iNOS+/+ and transplanted iNOS−/− mice with iNOS+/+ bone marrow but not in iNOS−/− animals. The loss of the iNOS gene in leukocytes of iNOS+/+ mice reduced iNOS mRNA expression by 59%. iNOS-deficient mice and iNOS+/+ animals with iNOS−/− leukocytes presented with a significant improvement in postoperative intestinal transit and in vitro smooth muscle contractility, whereas the replacement with iNOS+/+ bone marrow in iNOS−/− mice completely reversed this improvement. Conclusion:These results clearly show that iNOS expressed in leukocytes within the intestinal muscularis plays a major role in mediating smooth muscle dysfunction and subsequently postoperative ileus.

Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion

BACKGROUND & AIMS Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. METHODS We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14(+)HLA-DR(-/low) monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14(+)HLA-DR(-/low) cells by using blocking antibodies or knock-down technology. RESULTS Mature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14(+)HLA-DR(-/low) cells. Such CD14(+)HLA-DR(-/low) monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSC-induced development of CD14(+)HLA-DR(-/low) monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44. CONCLUSIONS Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver.

Endogenous endotoxin participates in causing a panenteric inflammatory ileus after colonic surgery

Objective:To investigate muscularis inflammation and endogenous endotoxin as causes of postoperative ileus. Background:Postoperative inflammatory ileus of the colon is associated with a significant delay in gastrointestinal transit. We investigated whether these changes are caused by the downstream obstructive barrier of the surgically altered colon or by small intestinal muscularis inflammation itself. Furthermore, we evaluated the mechanistic role of gut derived endotoxin in the development of postoperative intestinal dysfunction. Methods:Rats underwent surgical manipulation of the colon. Isolated gastrointestinal transit was analyzed in animals with ileostomy. The perioperative emigration of intracolonic particles was investigated by colonic luminal injection of fluorescently labeled LPS and microspheres. Mediator mRNA induction was quantified by real-time RT-PCR. Muscularis leukocytic infiltrates were characterized. In vitro circular muscle contractility was assessed in a standard organ bath. Results:Ileostomy rats presented with a significant delay in small intestinal transit after colonic manipulation. This was associated with leukocyte recruitment and inflammatory mediator mRNA induction within the small intestinal muscularis. Colonic manipulation caused the transference of intracolonic LPS and microspheres into the intestinal muscularis. Postoperative in vitro small intestinal circular muscle contractility was impaired by 42% compared with controls. Gut decontamination and TLR-4 deletion significantly alleviated the small intestinal muscularis inflammation and prevented intestinal muscle dysfunction. Conclusions:Selective colonic manipulation initiates a distant inflammatory response in the small intestinal muscularis that contributes to postoperative ileus. The data provide evidence that gut-derived bacterial products are mechanistically involved in the initiation of this remote inflammatory cascade.

Anti‐inflammatory role of glycine in reducing rodent postoperative inflammatory ileus

Background  Inflammatory events within the intestinal muscularis, including macrophage activation and leukocyte recruitment, have been demonstrated to participate in causing postoperative ileus. Recently, glycine has gained attention due to its beneficial immunomodulatory effects in transplantation, shock and sepsis.

Resident Macrophages are Involved in Intestinal Transplantation‐Associated Inflammation and Motoric Dysfunction of the Graft Muscularis

Gut manipulation and ischemia/reperfusion evoke an inflammatory response within the intestinal muscularis that contributes to dysmotility. We hypothesize that resident macrophages play a key role in initiating the inflammatory cascade. Isogenic small bowel transplantation was performed in Lewis rats. The impact of recovery of organs on muscularis inflammation was investigated by comparing cold whole‐body perfusion after versus prior to recovery. The role of macrophages was investigated by transplantation of macrophage‐depleted gut. Leukocytes were counted using muscularis whole mounts. Mediator expression was determined by real‐time RT‐PCR. Contractility was assessed in a standard organ bath. Both organ recovery and ischemia/reperfusion induced leukocyte recruitment and a significant upregulation in IL‐6, MCP‐1, ICAM‐1 and iNOS mRNAs. Although organ recovery in cold ischemia prevented early gene expression, peak expression was not changed by modification of the recovery technique. Compared to controls, transplanted animals showed a 65% decrease in smooth muscle contractility. In contrast, transplanted macrophage‐depleted isografts exhibited significant less leukocyte infiltration and only a 19% decrease in contractile activity. In summary, intestinal manipulation during recovery of organs initiates a functionally relevant inflammatory response within the intestinal muscularis that is massively intensified by the ischemia reperfusion injury. Resident muscularis macrophages participate in initiating this inflammatory response.

Liver sinusoidal endothelial cells contribute to CD8 T cell tolerance toward circulating carcinoembryonic antigen in mice.

Immunity against cancer is impeded by local mechanisms promoting development of tumor‐specific T cell tolerance, such as regulatory T cells, myeloid‐derived suppressor cells, or immunosuppressive factors in the tumor microenvironment. The release of soluble antigens, such as carcinoembryonic antigen (CEA) from colorectal carcinoma (CRC) cells, has been investigated for diagnostic purposes, but not for its immunological consequences. Here, we address the question of whether soluble CEA influences tumor‐specific immunity. Mice were injected with soluble CEA protein, and CEA‐specific CD8 T cells were analyzed for their phenotype and functionality by means of restimulation ex vivo or antitumor efficacy in vivo. We furthermore characterized the CD8 T cell population in peripheral blood mononuclear cell (PBMCs) from healthy donors and colorectal carcinoma patients. In mice, circulating CEA was preferentially taken up in a mannose receptor–dependent manner and cross‐presented by liver sinusoidal endothelial cells, but not dendritic cells, to CD8 T cells. Such systemically circulating CEA promoted tolerization of CEA‐specific CD8 T cells in the endogenous T cell repertoire through the coinhibitory molecule B7H1. These CD8 T cells were not deleted but were rendered nonresponsive to antigen‐specific stimulation and failed to control growth of CEA‐expressing tumor cells. These nonresponsive CD8 T cells were phenotypically similar to central memory T cells being CD44highCD62LhighCD25neg. We found T cells with a similar phenotype in PBMCs of healthy donors and at increased frequency also in patients with colorectal carcinoma. Conclusion: Our results provide evidence for the existence of an unrecognized tumor immune escape involving cross‐presentation of systemically circulating tumor antigens that may influence immunotherapy of cancer. (HEPATOLOGY 2012;56:1924–1933)

Role of resident macrophages in the immunologic response and smooth muscle dysfunction during acute allograft rejection after intestinal transplantation.

Resident muscularis macrophages initiate an inflammatory cascade during ischemia/reperfusion that is associated with dysmotility and the activation of immunologic processes. We hypothesized that these muscularis macrophages may also play a potential immunologic role for acute allograft rejection in intestinal transplantation. Orthotopic SBTx (BN‐Lew) was performed without immunosuppression. Animals were sacrificed 7 days after SBTx. The role of resident macrophages was evaluated by transplantation of macrophage‐depleted and gadolinium chloride‐treated gut. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by Real‐Time‐RT‐PCR. Apoptosis was evaluated by TUNEL. Smooth muscle contractility was assessed in a standard organ bath. In comparison to vehicle‐treated grafts, macrophage‐depleted grafts exhibited significantly lower mediator mRNA peak expression (IL‐6, IL‐2, IL‐10, MCP‐1, iNOS, TNFα, IFNγ, FasL), leukocyte infiltrates (ED1‐ and ED2 positive monocytes and macrophages, neutrophils, CD4+ and CD8+ lymphocytes), apoptosis rates and an improved histologic rejection grading. Vehicle‐treated grafts showed a 77% decrease in smooth muscle contractility compared to naïve controls, while macrophage‐depleted gut exhibited only a 51% decrease in contractile activity. Transplantation of macrophage‐depleted gut attenuates the functionally relevant molecular and cellular immunologic response within the graft muscularis in acute allograft rejection. Resident macrophages participate in initiating these processes.