Muscularis Macrophages Research Articles

Mechanism and Impact of Organ Harvesting and Ischemia-Reperfusion Injury Within the Graft Muscularis in Rat Small Bowel Transplantation

Introduction Inflammatory events within the gut muscularis contribute to dysmotility. We hypothesized that manipulation during organ harvesting initiated an inflammatory response via muscularis macrophages and that this cascade was amplified during reperfusion. Methods Small bowel transplantation was performed in Lewis rats. To investigate the impact of organ harvesting on muscularis inflammation, cold whole-body perfusion was performed after versus prior to organ harvesting. The role of macrophages was investigated by transplantation of the macrophage-depleted gut. Leukocyte infiltration was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase polymerase chain reaction. Contractility was assessed in a standard organ bath. Results Organ harvesting and ischemia-reperfusion induced leukocyte recruitment and mRNA upregulation in the muscularis: interleukin-6 12217-fold, monocyte chemoattractant protein-1 62-fold, ICAM-1 12-fold, cyclooxygenase-2: 8-fold, iNOS: 150-fold. Although organ harvesting with cold ischemia prevented early gene expression, peak expression at 3-hour reperfusion was not changed by modification of the harvesting technique. Compared to controls, transplanted animals showed a 63% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted gut exhibited significantly fewer leukocytes and only a 16% decrease in contractility. Conclusions Gut manipulation during organ harvesting initiates an inflammatory response within the muscularis that is massively intensified during reperfusion. This change contributes to muscular dysfunction. Furthermore, the results suggested that resident macrophages play a key role in initiating this process.

Depletion and inactivation of intestinal muscularis macrophages prevent postoperative ileus

Abstract 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.

Macrophages in the Small Intestinal Muscularis Externa of Embryos, Newborn and Adult Germ-Free Mice

Previously, we demonstrated the presence of a constant and regularly distributed macrophage population of ramified cells in the intestinal muscle layers of smaller rodents. The function of these resident macrophages under normal conditions remains unknown. Histochemistry, immunohistochemistry and electron microscopy were applied to the muscularis externa of 15- and 17-day-old embryos, 2-day-old mice, adult germ-free and conventional mice. Since lipopolysaccharides (LPS) activates macrophages and inflammation affects gut motility, LPS-treated mice were also included in the study. Two macrophage antibodies, F4/80 and 2F8 were used to demonstrate the presence of macrophages in the muscle layers. The localization was confirmed by electron microscopy. In contrast to conventional adult mice, the muscle layers in embryos, newborn and germ-free adult mice were devoid of class II MHC antigen reactive cells. The acid phosphatase reaction and antibodies directed towards a lysosomal protein (Lamp-2) were used in order to verify other activation markers. None of these showed specific staining of the muscularis macrophages. Only LPS-treated adult mice showed iNOS-positive cells in whole mounts. We conclude that the characteristic organization and distribution of muscularis macrophages in adult mice are also present in embryos, newborn and germ-free mice and thus develop independently of foreign antigens. Further, these macrophages are truly resident and appear to have differential responses to exogene stimuli.

Intra-abdominal activation of a local inflammatory response within the human muscularis externa during laparotomy.

To 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. Mild 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). Human 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-1beta, tumor necrosis factor (TNF)-alpha, 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. Normal 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-1beta, TNF-alpha, 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. These 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.

MCP-1 causes leukocyte recruitment and subsequently endotoxemic ileus in rat.

Endotoxemia causes an inflammatory response within the intestinal muscularis and gastrointestinal dysmotility. We hypothesize that the resident macrophage-derived chemokine monocyte chemoattractant protein-1 (MCP-1) plays a significant role in the recruitment of leukocytes into the lipopolysaccharide (LPS)-stimulated rat intestinal muscularis. MCP-1 mRNA expression was investigated by RT-PCR. Leukocyte extravasation and MCP-1 protein localization were determined by immunohistochemistry. Contractile activity was assessed by using a standard organ bath in rats that were treated with saline, recombinant MCP-1, LPS, LPS + nonspecific antibody, or LPS + MCP-1 antibody. Endotoxemia caused a significant 280-fold increase in MCP-1 mRNA expression in the muscularis, peaking at 3 h. MCP-1 protein was immunohistochemically located to muscularis macrophages. LPS application caused significant leukocyte recruitment into the muscularis and a 51% decrease in muscle contractility. MCP-1 antibody treatment significantly averted leukocyte recruitment and significantly prevented muscle dysfunction. These parameters were not significantly altered by the nonspecific antibody. Results show that resident muscularis macrophage-derived MCP-1 plays a major role in the recruitment of monocytes during endotoxemia, which then subsequently secrete kinetically active substances that cause ileus.

Prostanoid production via COX-2 as a causative mechanism of rodent postoperative ileus

This study demonstrates a significant role for cyclooxygenase (COX)-2 and prostanoid production as mechanisms for surgically induced postoperative ileus. Rats, COX-2+/+, and COX-2-/- mice underwent simple intestinal manipulation. Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize COX-2 expression. Prostaglandin levels were measured from serum, peritoneal lavage fluid, and muscularis culture media. Jejunal circular muscle contractions were measured in an organ bath, and gastrointestinal transit was measured in vivo. The data show that intestinal manipulation induces COX-2 messenger RNA and protein within resident muscularis macrophages, a discrete subpopulation of myenteric neurons and recruited monocytes. The manipulation-induced increase in COX-2 expression resulted in significantly elevated prostaglandin levels within the circulation and peritoneal cavity. The source of these prostanoids could be directly attributed to their release from the inflamed muscularis externa. As a consequence of the molecular up-regulation of COX-2, we observed a decrease in in vitro jejunal circular muscle contractility and gastrointestinal transit, both of which could be alleviated pharmacologically with selective COX-2 inhibition. These studies were corroborated with the use of COX-2-/- mice. Prostaglandins, through the induction of COX-2, are major participants in rodent postoperative ileus induced by intestinal manipulation.

Biphasic response to gut manipulation and temporal correlation of cellular infiltrates and muscle dysfunction in rat

Surgical manipulation of the intestine results in the massive movement of leukocytes into the intestinal muscularis at 24 hours. This is associated with muscle inhibition. The aim of this study was to temporally associate leukocyte extravasation with ileus after surgical manipulation. Rats underwent a simple manipulation of the small bowel and were killed at various times (0, 0.25, 0.5, 1, 3, 6, 12, and 24 hours) postoperatively. Jejunal circular-muscle contractile activity was assessed in a standard organ bath. Both extravasating and resident leukocytes were immunohistochemically stained in muscularis whole mounts. Contractile activity was significantly reduced immediately after surgery, but rapidly returned to control levels at 3 hours. After recovery, muscle function decreased at 12 and 24 hours (41% and 81%, respectively). The resident muscularis macrophage network demonstrated cellular activation 1 hour postoperatively. The number of leukocytes increased over time (neutrophils, 67.5-fold; monocytes, 98.2-fold; and mast cells, 47-fold at 24 hours). The functional results demonstrate a biphasic response in the suppression of muscle activity after surgical manipulation. Regression analysis (r2 = 0.998) of the temporal development of leukocyte infiltration and the protracted phase of muscle inhibition provides evidence for a correlation between cellular inflammation and postoperative dysmotility.

LPS-induced muscularis macrophage nitric oxide suppresses rat jejunal circular muscle activity.

Cellular mechanisms of sepsis-induced ileus remain an enigma. The study aim was to determine the role of nitric oxide (NO) in mediating the suppression of rat jejunal circular smooth muscle activity during endotoxemia. Isolated muscularis inducible NO synthase (iNOS) mRNA was measured by RT-PCR, immunohistochemistry was employed to localize iNOS protein, and contractile activity was measured in an organ bath. The low basal expression of muscularis iNOS mRNA expression was increased in a time-dependent fashion after lipopolysaccharide (LPS), resulting in a 20-fold increase over controls 3 h after injection. Immunohistochemistry of muscularis whole mounts and dissociated muscularis cells for iNOS revealed staining only in the muscularis macrophages 12 h after LPS. LPS caused a 68% reduction in spontaneous muscle activity 12 h after injection, which improved by 53% after the in vitro application of the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine. Similar results were obtained in C57BL/6 mice but not in iNOS knockout mice. These data demonstrate that macrophage iNOS plays an important role in mediating LPS-induced intestinal circular muscle suppression.

P–321 - Lipopolysaccharide inhibits intestinal smooth muscle contraction by inducing iNOS and COX-2 in the resident muscularis macrophage

P–321 - Lipopolysaccharide inhibits intestinal smooth muscle contraction by inducing iNOS and COX-2 in the resident muscularis macrophage

Leukocytes of the intestinal muscularis: their phenotype and isolation.

The basal presence of immunologically potent cells within the intestinal muscularis externa and their functional significance is unclear. Our aim was to investigate the basal distribution of various leukocyte populations within the rat jejunal muscularis. In addition, we sought to immunohistochemically phenotype the muscularis macrophage in jejunal whole-mounts, isolate these cells in primary culture, and investigate their ontogenesis. Macrophages form a regularly distributed network that expresses major histocompatibility complex class II, CD14 receptors, and a low level of CD11/CD18. The macrophages are activated by dissection and are present in fetal animals. Enriched macrophage cultures show a normal resident phenotype and remain present for weeks in dissociated muscularis cultures. The results also demonstrate the presence of neutrophils, monocytes, mast cells, and lymphocytes within the muscularis and suggest that the dense network of muscularis macrophages may be a potent resident trigger for inflammation in response to tissue injury or bacterial translocation.

Selective coinduction of cyclooxygenase-2 and inducible nitric oxide synthase in human intestinal smooth muscle during abdominal surgery

Postoperative ileus continues to be a major cause of prolonged hospitalization and morbidity after laparotomy. We have previously demonstrated that endotoxin-activation of the dense network of resident macrophages in the small bowel muscularis modulates muscle function through the rapid release of prostanoids and nitric oxide. Furthermore, in vitro activation of mouse macrophages by endotoxin has been shown to increase the inducible enzymes cyclonxygenase (COX-2) and nitric oxide synthase (iNnS). The aim of this study was to investigate the coinduction of these enzymes in the human gut after surgical manipulation. We obtained human small bowel specimens at two time points during common abdominal surgical procedures: less than 60 minutes and greater than three hours after incision. The mucosa and muscularis layers was isolated, rapidly frozen in liquid nitroge n and stored for RNA extraction. Semi-quantitative RT-PCR was used to measure COX-2 and iNnS mRNA levels in the separated layers. Fixed museularis whole-mounts were stained immunnhistochemically using monoclonal antibodies against COX-2, iNnS, macrnphages and the activation marker LFA-Ic~. Both inducible enzymes were strongly expressed in the separated layers at both time points. COX-2 and iNnS mRNA-levels were significantly increased in late compared to early specimens only in the muscularis (COX-2 3.4 fold, iNnS 2.2 fold, p<0.05), mucosal levels did not change. Immunohistochemistry showed a dense network of resident macrophages within the muscularis. LFA-la and iNnS positive cells were increased in late specimens, whereas early specimens were either negative or displayed only a weak signal. Morphologically, LFA-I~ and iNnS positive cells appeared to be macrophages. These results demonstrate that common abdominal surgical procedures activate resident macrophages in the muscularis extema and upregulate COX-2 and iNnS within human intestinal smooth muscle. The data suggest that the production of prnstaglandins and nitric oxide by muscularis macrophages could play a major role in the development of postoPerative ileus.

Lipopolysaccharide activates the muscularis macrophage network and suppresses circular smooth muscle activity.

Bacterial lipopolysaccharide (LPS) is a causative agent of sepsis-induced ileus. Although it is known that LPS activates macrophages and initiates inflammation, the consequences of LPS on the macrophage network and a potential inflammatory response within the intestinal muscularis have not been investigated. This study was designed to identify cellular and functional changes in rat intestinal muscularis after intraperitoneal LPS. Histo- and immunohistochemistry were used to phenotype leukocytes. Functional alterations were determined using an organ bath. Compared with controls, LPS caused a 21-fold increase in staining for the lymphocyte activation marker-1 (LFA-1) localized to the ED2+ macrophage network 1 h after injection. This was followed by a significant infiltration of neutrophils, mast cells, and monocytes into the muscularis. LPS also caused a 62% reduction in spontaneous circular muscle activity and a 91% suppression of bethanechol-stimulated contractions 12 h after injection. These results demonstrate that endotoxemia 1) acutely activates the muscularis macrophage network, 2) causes the extravasation of leukocytes, and 3) results in circular muscle impairment.