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Keywords:

  • barrier function;
  • c-Kit;
  • macrophages;
  • mast cells;
  • postoperative ileus

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References

Background  Abdominal surgery involving bowel manipulation commonly results in inflammation of the bowel wall, which leads to impaired intestinal motility and postoperative ileus (POI). Mast cells have shown to play a key role in the pathogenesis of POI in mouse models and human studies. We studied whether mast cells contribute to the pathogenesis of POI by eliciting intestinal barrier dysfunction.

Methods  C57BL/6 mice, and two mast cell-deficient mutant mice KitW/W-v, and KitW-sh/W-sh underwent laparotomy (L) or manipulation of the small bowel (IM). Postoperative inflammatory infiltrates and cytokine production were assessed. Epithelial barrier function was determined in Ussing chambers, by measuring transport of luminal particles to the vena mesenterica, and by assessing bacterial translocation.

Key Results  In WT mice, IM resulted in pro-inflammatory cytokine and chemokine production, and neutrophil extravasation to the manipulated bowel wall. This response to IM was reduced in mast cell-deficient mice. IM caused epithelial barrier dysfunction in WT mice, but not in the two mast cell-deficient strains. IM resulted in a decrease in mean arterial pressure in both WT and mast cell-deficient mice, indicating that impaired barrier function was not explained by tissue hypoperfusion, but involved mast cell mediators.

Conclusions & Inferences  Mast cell activation during abdominal surgery causes epithelial barrier dysfunction and inflammation of the muscularis externa of the bowel. The impairment of the epithelial barrier likely contributes to the pathogenesis of POI. Our data further underscore that mast cells are bona fide cellular targets to ameliorate POI.

Postoperative ileus (POI) is characterized by a transient cessation of intestinal motor activity following abdominal surgery, and as a result, patients suffer from complications and prolonged hospital stay.1,2 The costs related to POI have been estimated to amount 1.47 billion dollars annually in the USA, illustrating its large socio-economical impact.1 With regard to the pathogenesis of POI, it has become evident from animal and human studies that postoperative intestinal hypomotility in POI is the result of an influx of leukocytes into the manipulated muscularis externa.3,4 Neutrophil infiltrates have been shown to inhibit local contractile activity, i.e., via the release of nitric oxide (NO),5–7 or general motility via the activation of sympathetic inhibitory neural reflexes.8 The importance of this inflammatory response in POI is underscored, for instance, by the success of therapeutic strategies aimed at blocking neutrophil recruitment to ameliorate POI.1,3,9 However, the pathophysiological mechanisms behind the immune response to bowel manipulation remain to be clarified. In this respect, an important factor could be the reduced epithelial barrier function resulting from bowel handling that was previously observed in rodent models of POI.5,10 This would be in line with previous observations that bowel wall mechanical stretch11 and manipulation12 augments inflammatory responses of bowel wall macrophage populations and local dys-contractility via TLR activation.

We have previously shown that mast cells are crucial players in the intestinal inflammation that mediates POI 13 and that mast cell stabilizers and histamine receptor antagonists are instrumental in reducing POI in animal models13 and human POI.4,14 Mast cells are implicated in barrier dysfunction in animal models of chronic stress,15,16 allergic inflammation,17 parasitic infection,18 and endoxemia.19 Thus, given the implication of mast cells in the pathogenesis of POI, and their potential to regulate intestinal barrier function, we assessed the role of mast cell-induced barrier dysfunction in the occurrence of POI using two mast cell-deficient mouse strains, KitW/W-v and KitW-sh/W-sh. Here, we show that intestinal manipulation (IM), during abdominal surgery, is associated with intestinal barrier dysfunction and inflammation of the manipulated bowel muscularis externa. Our data indicate that both inflammation and barrier dysfunction are mediated by mast cells and can be considered as factors contributing to POI pathogenesis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References

Laboratory animals

Mice (C57BL/6, Harlan Nederland, Horst, The Netherlands) were kept under environmentally controlled conditions (light on from 8 AM to 8 PM; water and rodent non-purified diet ad libitum; 20–22 °C, 55% humidity). Mast cell deficient KitW/W-v (WBB6F1-W/Wv), the mast cell sufficient Kit controls (Kit+/+) and KitW-sh/W-sh (B6.Cg-KitW-sh/HNihrJaeBsmJ), and their C57BL/6 controls were from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were maintained at the animal facility of the Academic Medical Centre in Amsterdam and were used at 12–20 weeks of age. Animal experiments were performed in accordance with the guidelines of the Ethical Animal Research Committee of the University of Amsterdam.

Surgical procedures: abdominal surgery with intestinal manipulation

Mice were anesthetized by an intraperitoneal (IP) injection of a mixture of fentanyl citrate/fluanisone (Hypnorm; Janssen, Beerse, Belgium) and midazolam (Dormicum; Roche, Mijdrecht, The Netherlands). Surgery was performed under sterile conditions. Mice (8–11 per treatment group) underwent control surgery of only laparotomy (L) or laparotomy followed by IM as described earlier.13 After 24 h, mice were anesthetized and killed by cervical dislocation, mesenteric lymph nodes were harvested under aseptic conditions, and subsequently the small intestine was removed, flushed in ice-cold saline, divided into several segments and stored for further analysis.

FACS analyses

Mesenteric lymph nodes (MLN) were isolated at indicated time points after surgery and cleared from fat. Tissue was digested for 15 min using collagenase IV and cell suspension was obtained after filtering, and cells were resuspended, washed, and taken up in RPMI medium/10% FCS and incubated for 3 h in BrefeldinA. Cells were then washed and resuspended in FACS buffer, and incubated with the appropriate antibodies. Cells were fixed with 2% PFA; for intracellular FACS, cells were treated with 0.5% saponin in FACS buffer.

Bacterial translocation

Mesenteric lymph nodes were weighed, placed in a tube containing 300 μL of ice-cold Luria–Bertani (LB) broth, homogenized with a sterile grinder, and plated onto blood agar plates under aerobic and anaerobic conditions. After 48 h of incubation at 37 °C, the number of colony forming units (CFU) per milligram lymph node was assessed.

Ussing chamber experiments

Segments of tissue of the distal small intestine were opened, cut, and immersed in Modified Meyler’s Buffer (128 mmol L−1 NaCl, 4.7 mmol L−1 KCl, 1.3 mmol L−1 CaCl2 6H2O, 20.2 mmol L−1 NaHCO3, 0.4 NaH2PO4 H2O, 0.33 mmol L−1 Na2HPO4, and 1.0 MgCl2 6H2O, 10 mmol L−1 Hepes, 10 mmol L−1 glucose at pH 7.4). Ileal segments were removed 45 min after surgery and 15 min thereafter, the tissue was mounted in Ussing chambers (World Precision Instruments, Berlin, Germany). Serosal and mucosal areas were exposed to 2 mL of circulating oxygenated Modified Meyler Buffer maintained at 37 °C. After 15 min, horseradish peroxidase (HRP, Sigma-Aldrich, Steinheim, Germany) was added to the luminal buffer at a final concentration of 10 μmol L−1. After 30, 60, and 90 min, samples (300 μl) were taken from the serosal side and replaced with fresh buffer. The enzymatic activity of HRP was measured using o-Phenylenediamine dihydrochloride (Sigma-Aldrich) as a substrate. The transepithelial flux of HRP was represented as pmol h−1 cm−2.

In vivo intestinal permeability measurement

Mice (female C57BL/6 and KitW-sh/W-sh) were anesthetized using mixture of fentanyl citrate/fluanisone (Hypnorm; Janssen) and midazolam (Dormicum; Janssen). Abdominal surgery with intestinal manipulation was performed as described.8 Mice (= 5–7 per group) were assigned to the following two groups: laparotomy only (sham); laparotomy followed by IM. One hour after surgery, the mice were re-anaesthetized with Isoflurane, 1.2–2.5% vol (Abott Laboratories, Kent, UK) and cannulation of the superior mesenteric vein (SMV) was performed under sterile conditions. After ligating the ileum 5 cm from the ileocecal valve and 5 cm proximal from the initial ligation, 0.5 mL of saline solution containing fluorescent probe [Fitc-Dextran (3–5 kD, Sigma-Aldrich); Rhodamine Dextran (10 kD, Invitrogen, Carlsbad, CA, USA); in saline solution] was gently injected into the lumen. Blood samples (with concomitant fluid replacement) were drawn at intervals of 10 min until 1 hour from cannulation using Lithium heparin filled tubes (Greiner bio-one, Frickenhausen, Germany).

Antibiotic treatment

Gut decontamination was performed by administration of polymyxin B (75 mg kg−1 day−1) and neomycin (225 mg kg−1 day−1) as described earlier.12 The antibiotics were instilled into the stomach by a gastric tube for 6 days, once daily. Control animals received vehicle (normal saline) at appropriate time points. Mice underwent surgery with intestinal manipulation as described in the previous section on the sixth day of treatment and were killed on day 7.

Measurement of mean arterial pressure

Mean arterial pressure (MAP) was recorded as previously reported.20 In short, after induction of anesthesia, the carotid artery was cannulated and blood pressure and heart rate were recorded using a heparinized saline-filled catheter. The catheter was connected to a pressure transducer (Truwave PX-600F; Baxter, Deerfield, IL, USA) and signal samples and stored using Labview applications (National Instruments, Austin, TX, USA). Rectal temperature was monitored continuously and remained at 37 °C throughout the experiment. Sodium nitroprusside treatment: after being anesthetized using FFM, mice were injected with 50 μg kg−1 Sodium nitroprusside (SNP; Sigma-Aldrich) i.p. using an automatic infusion pump. The infusion rate was adjusted whenever necessary to maintain the desired MAP drop for the time indicated.

Gastrointestinal transit

Gastrointestinal function was determined in vivo by measurement of gastrointestinal transit of liquid FITC-dextran (70 000 D; Invitrogen, Paisley, UK). Ninety minutes after administration through oral gavage, the animal was killed and the entire bowel from stomach to distal colon was collected. The contents of the stomach, small bowel (divided into 10 segments of equal length), the cecum, and colon (three segments of equal length) were collected and assayed in duplicate for the presence of fluorescent label (Synergy HT, BioTek Instruments Inc., VT, USA; excitation wavelength: 485 nm, emission wavelength: 528 nm) for quantification of the fluorescent signal in each bowel segment. The distribution of signal along the gastrointestinal tract was determined by calculating the geometric center (GC): Σ (percent of total fluorescent signal in each segment × the segment number)/100 for quantitative statistical comparison among experimental groups. Individual transit distribution histograms were plotted, and transits were statistically analyzed using the calculated GC.

Mast cell culture and reconstitution of KitW-sh/W-sh mice

KitW-sh/W-sh mice were reconstituted by the injection of bone marrow-derived cultured mast cells into the peritoneal cavity, as described earlier.13 Sterile, endotoxin-free medium was flushed repeatedly through the bone shaft using a needle and syringe. The suspension of BM cells was centrifuged at 320 g for 10 min and cultured at a concentration of 0.5 × 106 nucleated cells mL−1 in RPMI 1640 with 10% FCS (Sigma-Aldrich) 100 units mL−1 penicillin, 100 μg mL−1 streptomycin (Life Technology, Breda, The Netherlands), 10 μg mL−1 gentamycine, 2 mmol L−1 L-glutamine, and 0.1 mmol L−1 non-essential amino acids (referred to as enriched medium) and a combination of IL-3 (5 ng mL−1) and SCF (50 ng mL−1) for 3 weeks at 37 °C in a humidified atmosphere with 5% CO2. Non-adherent cells were transferred to fresh medium at least once a week. After 3–4 weeks, when a mast cell purity of >95% was achieved, as assessed by toluidine blue staining, the cells were harvested for experiments. Three week old KitW-sh/W-sh received 10 × 106 cells in 100 μL PBS through IP injection. Mice were used 10 weeks after adoptive transfer of mast cells. This procedure reconstitutes the mast cell population without systemic effects.

Immunohistochemistry and visualization of myeloperoxidase positive cells

Immunohistochemical staining for CD11a and CD3 was performed on acetone fixed transverse ileal segments. Endogenous peroxidase activity was eliminated by incubation of segments in methanol containing 0.3% H2O2. Non-specific protein-binding sites were blocked by incubation in PBS, pH 7.4, containing 10% of normal goat serum for 10 min. Sections were incubated overnight with biotinylated hamster antimouse CD11a or CD3 antibodies (Pharmingen, San Diego, CA, USA.) (dilution 1 : 1000). Next, sections were incubated with ABComplex/HRP (DAKOCytomation, Glostrup, Denmark) for 30 min. HRP was visualized using SigmaFast DAB (Sigma-Aldrich), incubating 5 min, and couterstained with haematoxilin/eosin. Visualization and quantification of myeloperoxidase (MPO) positive cells in the ileal muscularis externa was performed as described elsewhere.21

Tissue cytokine production

For cytokine measurements, mucosa was separated from the muscle tissue using a glass slide, in icecold modified Meyler’s buffer. Six-cm segments were added to 500 μL lysis buffer containing 300 mmol L−1 NaCl, 30 mmol L−1 Tris, 2 mmol L−1 MgCl2, 2 mmol L−1 CaCl2, 1% Triton X-100; pepstatin A, leupeptin, and aprotinin (all 20 ng mL−1; pH 7.4), homogenized, and incubated at 4 °C for 30 min. Homogenates were centrifuged at 1500 × g at 4 °C for 15 min, and supernatants were stored at −20 °C until assays were performed. TNF, IL-6, MCP-1, RANTES, IL-12p70 and KC in supernatants were analyzed by mouse ELISA (R&D systems, Abingdon, UK) according to manufacturer’s instructions.

Statistical analysis

The data are expressed as mean ± SEM and were analyzed using the non-parametric Mann–Whitney U test. A P value less than 0.05 was considered significant. IM surgery groups were compared with L control groups where indicated.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References

Intestinal manipulation results in an immune response localized at the muscularis externa

We first established in a mouse model for POI that IM resulted in the activation and infiltration of CD11a-positive leukocytes in the muscularis externa of the small intestine. The intestinal muscularis is known to contain a network of resident macrophage-like cells of which a proportion is F4/80 positive (Fig. 1A).22 The activation of this cell population has been implicated in the leukocyte recruitment in POI3,13 leading to an extravasation of CD11a expressing cells that appeared in muscularis externa of the small intestine 24 h after IM (Fig. 1A,B). The population of CD11a positive cells is negative for CD3 as shown in consecutive sections (Fig. 1B) and comprises extravasated monocytes and neutrophils, as demonstrated earlier.3 We further assessed the IM-induced cytokine production 1 h, 3 h, 6 h, and 24 h after IM in the muscular layer and mucosa. IL-6 production and signaling is an important characteristic of POI induced inflammation23 and hence, we choose to measure IL-6 along with chemokines MCP-1 (CCL-2) and KC (the mouse homologue of IL-8) as key parameters of localized inflammation. In 3–24 h after IM, we observed a significant increased production of the cytokines CCL-2, IL-6, and KC in the mice that had undergone IM, but not in control L mice (Fig. 1C, left panels); levels of IL-12p70 and TNF were not significantly induced within this time frame. In conjunction with previous reports,24 the IM procedure did not lead to significant cytokine production in the small intestinal mucosa (Fig. 1C, right panels). Notably, RANTES was constitutively expressed in the mucosa and unaffected by IM (Fig. 1C).

image

Figure 1.  Intestinal manipulation (IM)-induced inflammation is localized to the muscularis externa. In animals that underwent laparotomy (L), CD11a-expression, indicating leukocyte activation, is absent from the muscularis externa (A, left panel). Resident macrophages are present in the muscularis externa of unmanipulated mice, as indicated by F4/80 staining (A, right panel). After IM, CD11a expression in the muscularis externa was induced (B, right panel). T-cells (CD3+) do not infiltrate the muscularis externa 24 h after IM (B, right panel). IM-induced production of cytokines and chemokines in muscularis externa homogenates (C, left panels) and in mucosa homogenates (C, right panels) is shown at indicated time points after surgery. (C). RANTES protein expression was unaltered (C, left panels) and constitutively expressed in the small intestinal mucosa (C, right panels); *< 0.05, **< 0.01 (compared with the respective L groups). Bars indicate mean ± SEM. (= 6 per group).

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In a recent study, intestinal surgery and manipulation was reported to elicit DC derived IL-12 production within 30 min after surgery, initiating a Th1 response leading to POI.25 In our model, enhanced production of IL-12 at earlier time points after IM in the muscularis tissue was not apparent and IL-12 increased in muscle tissue after 24 h (Fig 1C), i.e., at a time point where an inflammatory infiltrate was already established.

IM-induced inflammation is dependent on the presence of mast cells

We have previously implicated mast cells in the pathogenesis of POI. Hence, we sought to establish that mast cell activation is involved in the muscularis inflammatory response and POI after intestinal surgery. Upon activation, connective tissue mast cells release chymases such as mouse mast cell protease-1 (mMCP-1). MMCP-1 release in peritoneal lavage fluid was increased 1 hour after intestinal manipulation compared with L (Fig. 2A). In C57BL/6 mice, mast cells were found in the muscularis externa, Peyer’s Patches, and mesentery (Fig 2A). Next, we investigated the role of mast cells in the leukocyte recruitment to the ileum muscularis externa observed after IM. To this end, we studied the response to IM in two mast cell-deficient mice strains that carry different spontaneous mutations in the gene for ckit [White spotting (W) locus] KitW/W-v and KitW-sh/W-sh mice. Reduced c-kit tyrosine kinase-dependent signaling results in mast cell reduction in both mouse strains. In KitW/W-v mice, a reduced inflammatory infiltrate was observed after IM if compared with their mast cell-sufficient Kit+/+ cognate controls (Fig 2B). In addition, the production of cytokines MCP-1 and IL-6 depends on the appropriate development of mast cells as both cytokines are significantly reduced in KitW/W-v mice (Fig. 2B). These data were corroborated by a reduced cellular MPO positive infiltrate, and reduced levels of IL-6 and MCP-1 after IM in mice that were treated with mast cell stabilizers ketotifen or doxantrazole (13 and data not shown).

image

Figure 2.  Mast cells are crucial in muscularis externa inflammation following bowel handling. Mouse mast cell protease (mMCP-1), determined in peritoneal lavage 1 h after surgery, was increased after intestinal manipulation (IM) compared with laparotomy (L) (A). Mast cells (purple) were visualized in C57BL/6 mice with Toluidin Blue staining and were present muscularis externa, serosa, Peyer’s patches and the mesentery (A). The number of myeloperoxidase (MPO) positive cells per mm2 and production of pro-inflammatory cytokines MCP-1 and IL-6 in the muscularis externa was significantly reduced in mast cell-deficient KitW/W-v compared with control mice (Kit+/+) (B). In KitW-sh/W-sh mice, the number of MPO+ cells was not increased after IM compared with control (C57BL/6). Similarly, in KitW-sh/W-sh reconstituted with C57BL/6 bone marrow derived mast cells, the number of MPO positive cells as unaffected after IM (C). CCL-2, and IL-6 production in KitW-sh/W-sh and C57BL/6 and KitW-sh/W-sh reconstituted with bone marrow derived mast cells (D). Asterisks indicate significant differences where indicated; **< 0.01, *< 0.05. Bars indicate mean ± SEM, for all groups L: = 5–7 IM: = 10 mice per group.

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In addition to a mast cell deficiency, in KitW/W-v mice, haematopoiesis is affected, they are anemic and infertile.26,27 Therefore we additionally analyzed the alternative c-kit mutant KitW-sh/W-sh and the respective WT strain C57BL/6, that comprise a more selective mast cell-deficient phenotype compared with the KitW/W-v strain.27

In contrast to C57BL/6 controls, IM in the KitW-sh/W-sh mice did not lead to a significant increase in MPO positive cells compared with controls (Fig. 2C). The production of IL-6 and CCL-2 in their muscular compartment after IM surgery was significantly reduced compared with their C57BL/6 controls (Fig. 2D). To demonstrate that the observed phenotype was mast cell dependent, we reconstituted KitW-sh/W-sh mice with mast cells (MC) obtained from C57BL/6 bone marrow derived mast cell cultures, and measured the cytokine response to IM. Success of reconstitution was demonstrated by presence of mast cells in KitW-sh/W-sh ileum (see Fig 3C). From these analyses, we concluded that the number, or tissue location, of ileal mast cells found in mast cell reconstituted KitW-sh/W-sh mice was not different from those found in C57Bl/6 mice (Fig 3C). In addition, earlier studies have shown that this treatment allows reconstitution to functionally active mast cells.13,27 We next assessed whether mast cell reconstitution restores the inflammatory response to IM. In contrast to vehicle treated KitW-sh/W-sh mice, in KitW-sh/W-sh mice that were reconstituted with C57Bl/6 cultured mast cells, the production of CCL-2 after IM was significantly increased, and was comparable to C57BL/6 WT levels. IM induced IL-6 (Fig. 2D) was unaffected by reconstitution (Fig. 2D), indicating that mast cells do not play a role in the induction of IL-6 in the model.

image

Figure 3.  Role of mast cells in gastrointestinal transit after intestinal manipulation. Twenty four hours after laparotomy (L) or intestinal manipulation (IM), gastrointestinal transit was determined by the % distribution of the fluorescent marker and calculation of the geometric center (GC) as indicated in materials and methods. St = stomach, cc = cecum. The fluorescent marker (dextran-FITC) in the L groups is located more proximally in the KitW-sh/W-sh than in the C57BL/6 mice as depicted in (A, left panel). After IM, dextran is localized more proximally in C57BL/6 mice than in KitW-sh/W-sh mice (A, middle panel). IM-induced delay in gastrointestinal transit is partially rescued by reconstitution of KitW-sh/W-sh with WT mast cells (A, right panel). GC values are shown in (B). *< 0.05 (compared with the respective L group). Bars indicate mean ± SEM. (L: = 4, IM: = 4–7 mice per group). (Panel C) Mast cells were visualized using toluidine blue staining in KitW-sh/W-sh mice (upper panel), or KitW-sh/W-sh mice that were reconstituted with C57BL/6 cultured mast cells (lower panel). (D) the quantification of the number of mast cells recovered in the ileum after reconstitution. N = 4 mice per treatment group. ND is not detectable.

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IM-induced delay in gastrointestinal transit is dependent on the presence of mast cells

We next examined the role of mast cells in POI by analyzing the functional changes in gastrointestinal transit induced by IM. We choose for KitW-sh/W-sh mouse strain to study the effect of mast cell activation on motility, because the use of KitW-sh/W-sh mice allows the motility parameters to be compared with C57BL/6 mice as these mice are derived on a C57BL/6 background. In the L control group, 24 h after surgery, gastrointestinal transit in KitW-sh/W-sh mice was delayed compared with C57BL/6 control mice. The majority of the fluorescent marker in KitW-sh/W-sh mice is present in segment (sg)7 and sg8, whereas the marker in the C57BL/6 mice is mainly present in sg9, sg10, and the cecum (Fig. 3A, left panel). This is reflected in the average calculated GC (Fig. 3B). After IM, in C57BL/6 mice, the gastrointestinal transit is significantly delayed as the fluorescence marker is mainly present in sg4 and sg5 (Fig. 3A, middle panel), also shown by the GC (Fig. 3B). However, the KitW-sh/W-sh mice display only a minor delay in gastrointestinal transit after IM (Fig. 3A, middle panel), clearly reflected in the GC values shown in Fig. 4B. After KitW-sh/W-sh mice were reconstituted with WT mast cells, the delay in gastrointestinal transit partially returned, indicated by the more widespread distribution of the fluorescent marker, in sg2, sg3, sg5, and also sg10 (Fig 3A, right panel). The IM-induced delay is also reflected in decreased GC values (Fig 3B). A significant decrease in GC value was observed after IM in control C57BL/6 mice, but not in KitW-sh/W-sh. Although mast cell reconstitution in these mice partially protects from POI as judged by the transit data (Fig. 3A), the GC values were unaffected and were not reduced as seen in C57BL/6 WT mice.

image

Figure 4.  Role of mast cells in IM-induced bacterial translocation. Bacterial translocation to the mesenteric lymph nodes (MLN) was determined 24 h after bowel handling. Following intestinal manipulation (IM), translocation of both aerobic and anaerobic cultured bacteria was significantly increased in C57BL/6 mice as compared with laparotomy (L) controls (L: = 5, IM: = 10 mice per group) (A). Bacterial translocation was also increased after IM in Kit+/+ mice and KitW/W-v mice compared with the L groups but did not differ between the two groups (B). The number of CFU cultured from mesenteric lymph nodes of KitW-sh/W-sh mice was not significantly different between L and IM groups (C). KitW/W-v and Kit+/+: L: = 5, IM: = 10; KitW-sh/W-sh and C57BL/6: L: = 3, IM: = 7 mice per group. Asterisks *< 0.05 (compared with L group), bars indicate mean ± SEM.

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IM induces mast cell dependent bacterial translocation to MLN

Given the implication of mast cells in epithelial barrier dysfunction in other models, we next assessed whether mast cells may contribute to the pathogenesis of POI by affecting the epithelial barrier function. To this end, we analyzed barrier function after IM in three ways: by determining postoperative bacterial translocation to draining mesenteric lymph nodes (MLN), by measuring permeability to HRP in Ussing chambers, and by assessing postoperative particle leakage from the small intestinal lumen into the blood circulation. We first assessed whether IM led to bacterial translocation 24 h after surgery by assessing the number of CFU in the MLN. In C57BL/6 mice that had undergone IM, the number of bacteria that had translocated to the MLN was significantly increased as compared with the L control group (Fig. 4A). The numbers of CFU cultured from MLN under aerobic and anaerobic culture conditions were comparable and therefore, in subsequent experiments, we only cultured bacteria under aerobic conditions. We next assessed whether mast cells play a causal role in the impaired intestinal barrier function after IM. To this end, we measured bacterial translocation in mast cell-deficient KitW/Wv mice. Baseline and IM-induced bacterial translocation did not differ in KitW/W-v mice, but this was not a characteristic of the lack of mast cells per se as the number of CFU was also not different in the Kit+/+ control mice (Fig. 4B). In the KitW-sh/W-sh mice, the IM-induced bacterial translocation was not increased after IM as compared with C57BL/6 control mice, although it should be noted that the actual number of translocating bacteria is generally low in these mice (Fig. 4C). Irrespectively, these data indicate that mast cells are likely to play a role in the bacterial translocation to the MLN after intestinal surgery.

Intestinal manipulation leads to an impaired barrier function via mast cell activity

The number of viable bacteria cultured from the MLN is dependent on the integrity of the epithelium, but also largely depends on processes such as uptake, and killing capacity CX3CR1-positive dendritic cells and macrophages.28 To specify the effect of IM on barrier function, we therefore decided to additionally assess epithelial permeability for protein directly after the IM surgery in Ussing chambers. We measured flux of HRP in ileum segments mounted 1 h after L or IM. In the IM group, the mucosal to serosal flux of HRP was enhanced as compared with the L group (Fig. 5A), reflecting an increase in epithelial permeability after IM. In contrast, however, ileal permeability measured in Ussing chambers was not significantly enhanced by IM in mast cell-deficient mice KitW-sh/W-sh (Fig 5A), KitW/W-v, and Kit+/+ (Fig 5B). To further substantiate these data, we additionally measured intestinal permeability for different sized luminal particles in vivo 1 h after IM surgery, i.e., at a time point at which no neutrophil recruitment was observed yet. To this end, we assessed transport of luminal dextran particles to the draining vena mesenteric vein. We observed a significant increase in the concentration of dextrans of 3–5 kD (Fig 5C, left panel) as well as 10 kD (Fig. 5C, right panel) in mesenteric venous blood 1–2 h after IM, but not after L. As unaffected tight junctions only allow transport of particles smaller than about 700 D, leakage of particles of a bigger size suggest a disarrangement of tight junction proteins resulting from IM. In conjunction, in vivo measurement of intestinal permeability using luminal 3–5 kD and 10 kD dextran particles demonstrated that in KitW-sh/W-sh mice were completely protected against IM induced epithelial permeability changes (Fig 5D). Together, these data indicate that mast cells are required for the pathological barrier dysfunction, associated with inflammatory response to the muscle layer and POI.

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Figure 5.  Mast cells regulate the transepithelial flux of small particles after intestinal manipulation. Mucosal to serosal transfer of horse radish peroxidase (HRP) was determined in Ussing chambers 1 h after surgery in ileal tissue of C57BL/6 and mast cell-deficient KitW-sh/W-sh and KitW/W-v. The mucosal to serosal flux of HRP was increased after intestinal manipulation (IM) compared to laparotomy (L) (A) in C57BL/6, but not in the mast cell-deficient KitW-sh/W-sh (A) and KitW/W-v (B) (= 5 mice per group). In C57BL/6, the flux of 3-5 kD (C, left panel) and 10 kD (C, right panel) particles from the small intestinal lumen to mesenteric blood is shown at indicated timepoints 1 h after IM (open circles) as compared with sham (closed circles). In KitW-sh/W-sh, the IM-induced increase in transepithelial flux particles was completely abolished as indicated in D (left panel: 3 kD, right panel: 10 kD). Asterisks *< 0.05 (compared with L group (A)) **< 0.01, ***< 0.001 [compared with sham-group (C)], bars indicate mean ± SEM. (= 5 mice per group).

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We next assessed whether bacterial translocation would play a significant role in the pathogenesis of POI. To study this, we decontaminated the gut using a 6-day regiment of antibiotic treatment and examined the effect of gut decontamination in our model of POI. As described earlier for colonic ileus,12 decontamination reduced the number of infiltrating leukocytes significantly (Fig 6A) and improved postoperative transit after IM (Fig. 6B). These data indicate a role for intestinal bacteria in the pathogenesis of POI and point towards possibility that enhanced bacterial translocation could contribute to the inflammatory response and POI after IM.

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Figure 6.  Antibiotics treatment reduce inflammatory parameters and improve postoperative transit. (A) Antibiotics treatment reduces IM induced inflammatory infiltrates in the muscularis externa, and improves postoperative transit (B-left panel) reflected by an enhanced GC value (panel B, right). N = 4, data shown are average ± SEM. Asterisks *P < 0.05 [compared with saline treated-group (A)] and **< 0.01 [compared with L group (B)].

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Intestinal manipulation induced decrease in blood pressure does not account for barrier dysfunction

To assess whether mast cells contribute directly to the decreased barrier function in our POI model, or whether that involves alternative mechanisms, we next explored other mechanisms that lead to barrier dysfunction, such as intestinal hypoperfusion.29,30 To this end, we next assessed whether IM coincided with a decrease in MAP, by monitoring MAP in arterial cannulated mice during abdominal surgery. As shown in Fig. 7, after opening the abdomen, the MAP was significantly reduced, whereas the reduction in the L group was minimal (Fig. 7A). This effect was independent of mast cells as in the KitW-sh/W-sh, IM led to a similar decrease in blood pressure if compared with WT (Fig. 7B). For both mouse strains, the decreased MAP sustained until mice recovered from anesthesia after 40 min (Fig. 7A/B right panels), whereas no difference in heart rate between L and IM was observed (results not shown). To validate whether the decrease in MAP alone could account for the impaired barrier dysfunction, we administered SNP, an NO donor, to achieve a decrease in MAP comparable to that seen after IM surgery. Intravenous rate-controlled perfusion of SNP allowed dosing SNP to lower MAP to mimic that observed after IM (Fig. 7C). SNP treatment did not trigger an inflammatory reaction 24 h after administration (Fig 7D, nor did it lead to bacterial translocation to MLN (Fig 7E), or epithelial barrier dysfunction (Fig. 7F). From these data, it can be concluded that the reduced MAP during IM does not account for the impaired barrier function, or the inflammatory response after IM, and that mast cells are involved in the pathogenesis of POI via a mechanism that is independent of the reduction in MAP.

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Figure 7.  Blood pressure during IM procedure. Mean arterial blood pressure (MAP) was recorded during the surgical procedure. Surgery was started when blood pressure was stabile (t = 0). At t = 5′, the % of MAP as compared with MAP at t = 0 (basal level) was significantly decreased during intestinal manipulation (IM) but not laparatomy (L) in both C57BL/6 (A, left panel) and mast cell-deficient KitW-sh/W-sh mice (B, left panel). Representative MAP recording during surgery is shown in right panels of A (C57BL/6) and B (KitW-sh/W-sh) (L = grey line) and IM (black line). Absolute values are shown at at the top of the graphs (A and B) (= 3 mice per group). Administration of the blood pressure lowering agent sodium nitroprusside (SNP) gives similar blood pressure reduction as compared with IM (C). SNP administration does not affect MPO count (D), bacterial translocation (E) 24 h after IM or epithelial permeability (F) at indicated time points 1 h after IM. Asterisks *P < 0.05 (compared with L-group), bars indicate mean ± SEM. (control: = 4; SNP: = 3 mice per group).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References

The prolonged impairment of gastrointestinal motility after intestinal manipulation is a significant confounding factor in postoperative recovery. Rodent models and human studies have demonstrated that surgical inspection and manipulation of the bowel leads to the activation of antigen presenting cells that reside in the intestinal muscularis layer.1,25 The general paralysis of the entire GI tract – including the unmanipulated segments – is a commonly seen characteristic of POI. This clinically important aspect of POI involves the activation of an inhibitory neural reflex arch by local inflammatory infiltrates,8 and was recently also shown to involve the production of IFN-γ by CCR9 + T-cells that are activated at the site of manipulation.25 We have shown previously that the activation of mast cells resulting from local manipulation of the bowel is a pivotal factor in the pathogenesis of POI and the inflammatory response to local manipulation.4,13,14 Hence, we questioned in the current study whether mast cell derived mediators contribute to POI either as local activators of dendritic cells recruited to the gut wall or via epithelial permeability changes.

We show here that intestinal manipulation induces barrier dysfunction via a mechanism that is crucially dependent on mast cells. In patients, barrier dysfunction frequently occurs during abdominal surgery and has been associated with increased postoperative septic morbidity in surgical patients undergoing laparotomy.31–33 In addition to this model of POI,13 as well as in human ileus,4 mast cell activation has been associated with disturbed intestinal barrier function in ulcerative colitis34 and several disease entities such as stress-induced hypersensitivity of the bowel15 and endotoxemia.19 In these models, the rapid release of serine proteases following triggering of mast cells,15 possibly via release of corticotropin releasing factor (CRF),34,35 is responsible for an increase in epithelial permeability. Likewise, the nature of the mast cell mediators that affect barrier function in our model involves similar rapid mechanisms and mediator release. This mechanism may involve the activation of protease activated receptor-2 (PAR-2) that is expressed on epithelial cells,36 although most studies have focused on PAR-2 activation by protease activity in the lumen.

Our data are in line with earlier observations in patients and rodent models implicating a disturbed intestinal barrier function after intestinal surgery involving bowel handling.37,38 It has been shown that in POI, bacterial products may reach the intestinal muscularis after IM of the small bowel,38 and that antibiotic treatment decreases muscular inflammation after colon manipulation.12 The implications of this process for the pathogenesis of POI are incompletely understood, but irrespective thereof, the clinical impact of bacterial translocation during surgery is significant. A recent study which included 927 patients over 13 years showed that bacterial translocation was associated with increased postoperative septic morbidity in surgical patients undergoing laparotomy.31

In this study, we assessed barrier integrity by measuring bacterial translocation to the MLN. Most probably this process reflects dendritic cell39 (or CX3CR1 expressing macrophages40) – mediated uptake of bacteria that are still viable once transported by dendritic cells to the MLN. Thus, the bacterial translocation is dependent on a number of immunological processes including phagocytosis, killing and bacterial cultures in the MLN may not reflect merely epithelial integrity. Therefore, we performed measurements of barrier function in the small intestine in Ussing chambers, reflecting the para- and transepithelial transport of the 40 kD HRP, as well as in vivo measurement of real time changes in epithelial leakage to the vena mesenteria. Using this combination of methods, we demonstrated that IM in our model of POI led to a mast cell dependent epithelial barrier dysfunction. This mechanism may explain the important role of mast cells in the pathogenesis of POI and validate mast cells as a bona fide drug target to shorten POI and improve postoperative recovery and barrier function. We performed our experiments in two strains of mast cell-deficient mice, KitW/W-v, and KitW-sh/W-sh mice, both of which carry mutations in the Kit gene [White spotting (W) locus]. C-kit is the receptor for stem cell factor (SCF) and involved in regulation of hematopoiesis, proliferation and migration of primordial germ cells and melanoblasts during development. The KitW/W-v mice carry the W mutation, resulting in deletion of the transmembrane domain of the c-kit protein as well as the dominant negative Wv mutation, a point mutation that affects c-kit kinase activity. On the other hand, the mast cell-deficient Kit-w-sh/w-sh mice carry a mutation that reflects an inversion in the kitlocus spanning a 2.8 mb segment. Hence, the resulting phenotypes are different: Kitw-wv mice have phenotypic abnormalities including sterility, anemia, lack of interstitial cells of Cajal (ICC), and have defects in hematopoiesis that lead to an absence of intra epithelial T-cells, neutropenia, and poor mobilization of blood neutrophils.26,41,42 In contrast, Kit-w-sh/w-sh mice, bearing the W-sash [W(sh)] inversion mutation, have mast cell deficiency, but are neither anemic nor sterile. Adult Kit-w-sh/w-sh mice have been shown to have a profound deficiency in tissue mast cells but normal levels of major classes of other differentiated hematopoietic and lymphoid cells.27

As the pathogenesis of POI is mediated by neutrophil influx, we reasoned that the protection after IM in our POI model in these KitW/W-v mice might not be selectively dependent on the lack of mast cells. Therefore, we also carried out our experiments in the KitW-sh/W-sh mice for two reasons: first, in contrast to the KitW/W-v, the KitW-sh/W-sh mice can be tested against C57BL/6 WT control mice. Second, KitW-sh/W-sh have normal neutrophil numbers, are fertile, contain normal number of intra epithelial lymphocytes (IELS), and are not anemic. The latter probably explains why the effects of IM-induced neutrophilic extravasation and inflammatory response were much less pronounced in the KitW-sh/W-sh compared with the KitW/W-v.

Interstitial cells of Cajal (ICC) require intact ckit signaling for proper development and are defective in both ckit mutant mice, although ICC subsets have been shown to develop in KitW-sh/W-sh mice.43 As KitW/W-v mice are hemizygous, we assessed basic GI motility in KitW-sh/W-sh that are on a C57BL/6 background. Indeed, we observed a disturbed motility and delayed transit under normal conditions compared with C57BL/6 mice. However, this difference did not reach significance and hence, we decided to include KitW-sh/W-sh in our motility analyses.

The observation that the bacterial translocation in KitW/W-v was almost completely abolished in both affected KitW/W-v and its control Kit+/+ may reflect a defective representation of innate immune cells in the lamina propria in these mice, given the purported role of lamina propria APCs in bacterial sampling.39,44 In addition, we observed that inflammatory parameters, bacterial translocation, and permeability to HRP differed between the control groups: C57BL/6 and Kit+/+. This is probably due to differences in immune responses that generally exist between mouse strains: for example between Balb/c and C57BL/6.45

In this study, we found that luminal bacteria were involved in IM-induced inflammation and ileus by treating mice with antibiotics, indicating that bacterial translocation contributes to the POI. It has been described that the immune response is induced soon after IM, for example ICAM-1 mRNA is expressed in the muscularis within 15 min of manipulation,3 whereas luminal products start to appear in the muscularis externa externa 6 h after intestinal manipulation,38 indicating that translocated bacterial antigens may not trigger muscularis immune responses, but may exacerbate immune responses, as shown by our data presented here. It is important to note that lamina propria intestinal macrophages that form the first line of defense generally do not produce high levels of cytokines upon bacterial challenge. Rather, dendritic cells in draining lymph are activated by luminal antigens which may not lead to mucosal inflammation but can contribute to the pathogenesis of POI in unmanipulated areas.25

We show in this study that IM is associated with a postoperative decrease in MAP. Hypoperfusion of intestinal tissue following abdominal as well as non-abdominal surgery has been associated with impaired barrier function preceded by hypotension, mesentery hypoperfusion and enterocyte damage.29,46 In addition, these studies show that aberrations in actin reorganization, cell proliferation and mitochondrial function are maximal at 60 min after mechanical bowel manipulation (i.e., the same time point at which we measured intestinal barrier function in the current study) and was partially recovered after 24 h46,47 (at which we measured inflammatory mediators). Although MAP decreases after IM, when MAP was pharmacologically lowered, the intestinal barrier function was not affected and inflammation did not occur to a similar extent. Circulating SNP-derived NO causes smooth muscle relaxation and subsequent microvascular vasodilatation. NO might affect inflammatory processes and intestinal barrier48,49 function independent of blood pressure alterations, but we show that NO has no effect on these processes. Of note, MAP measured in the carotid artery in our study probably reflects the blood pressure of the internal organs, but we cannot exclude that perfusion in the small intestine is different from the carotid artery.

Concluding, we show that IM elicits a mast cell dependent inflammatory response and intestinal barrier disturbances that may contribute to the pathogenesis of POI. Our study further underscores the potential of mast cell stabilization in ameliorating postoperative recovery, warranting that this treatment strategy should be pursued in clinical setting.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References

The authors are thankful for grant support from TIPharma, grant 1-215 to SAS, SAvD, JMD and RvdW. Further grant support is acknowledged from the Dutch Organization for Scientific Research (NWO; a VICI grant to CAC, GEB and SWvB and a VIDI grant to SD and WdJ). GEB was supported by a grant (Odysseus program, G.0905.07) of the Flemish “Fonds Wetenschappelijk Onderzoek” (FWO). SAS was supported by the Dutch organization for digestive diseases (MLDS, 05-45).

Author Contributions

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References

SAS, SD, SHB, JMD, SAD, OIS, AG, and FWH performed the research; CJZ, RMW, WJJ and GEB designed the study, analyzed data, and contributed essential reagents or tools; WJJ and SAS analyzed the data and wrote the paper.

Conflict of Interests

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References

No conflict of interest exists that could be perceived to bias the work.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Author Contributions
  8. Conflict of Interests
  9. References