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NEUROREGULATION AND MOTILITY

Mechanisms of polymicrobial sepsis-induced ileus

¹Department of Medicine, Division of Gastroenterology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15261; and ²Klinik und Poliklinik für Allgemein-, Viszeral-, Thorax- und Gefäßchirurgie, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn 53105, Germany

Submitted 20 August 2003 ; accepted in final form 8 April 2004

	ABSTRACT

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Sepsis frequently occurs after hemorrhage, trauma, burn, or abdominal surgery and is a leading cause of morbidity and mortality in severely ill patients. We performed experiments to delineate intestinal molecular and functional motility consequences of polymicrobial sepsis in the clinically relevant cecal ligation and puncture (CLP) sepsis model. CLP was performed on male Sprague-Dawley rats. Gastrointestinal transit, colonic in vivo pressure recordings, and in vitro muscle contractions were recorded. Histochemistry was performed for macrophages, monocytes, and neutrophils. Inflammatory gene expressions were quantified by real-time RT-PCR. CLP delayed gastrointestinal transit, decreased colonic pressures, and suppressed in vivo circular muscle contractility of the jejunum and colon over a 4-day period. A leukocytic infiltrate of monocytes and neutrophils developed over 24 h. Real-time RT-PCR demonstrated a significant temporal elevation in IL-6, IL-1, monocyte chemoattractant protein-1, and inducible nitric oxide synthase, with higher expression levels of IL-6 and inducible nitric oxide synthase in colonic extracts compared with small intestine. Polymicrobial CLP sepsis induces a complex inflammatory response within the intestinal muscularis with the recruitment of leukocytes and elaboration of mediators that inhibit intestinal muscle function. Differences were elucidated between endotoxin and CLP models of sepsis, as well as a heterogeneous regional response of the gastrointestinal tract to CLP. Thus the intestine is not only a source of bacteremia but also an important target of bacterial products with major functional consequences to intestinal motility and the generation of cytokines, which participate in the development of multiple organ failure.

inducible nitric oxide synthase; cecal ligation and puncture; lipopolysaccharide; proinflammatory cytokines

Exogenous lipopolysaccharide (LPS) from gram-negative bacteria is known to be a causative factor of sepsis-induced ileus, and the use of LPS as a selective stimulus for ileus is mechanistically valuable (3, 6, 7, 12, 19). It has previously been shown that LPS initiates an inflammatory cascade of events that consists of the activation of the normally quiescent network of resident intestinal muscularis macrophages (6, 28) and the elaboration of a plethora of inflammatory cytokines, chemokines, and substances (nitric oxide and prostaglandins) from the muscularis externa. This inflammatory milieu subsequently results in the recruitment of circulating leukocytes (33) and thus the further release of leukocyte-derived substances. Many of these factors, such as nitric oxide and prostaglandins, are known to mechanistically be involved in causing ileus through their direct alteration of the kinetic properties of intestinal smooth muscle cells (22, 30). Additionally, a strong case can be made for the influence of LPS-induced cytokines (IL-6, IL-1, and tumor necrosis factor-) in altering enteric neuromuscular transmission as key factors during sepsis (2, 10, 19).

Although much has been learned from the LPS injection models, they have been criticized for various reasons. Typically, exogenous LPS produces serum cytokine responses that are transient and are of a much greater magnitude than those observed in septic patients. LPS also causes a severe hypodynamic circulatory response without the initial hyperdynamic circulatory response as is seen clinically. Additionally, pharmacological agents and manipulations that have been shown to be effective in exogenous LPS animal models have failed in clinical trials (4, 9, 26). For instance, calcium antagonists improve endotoxin survival, but they increase mortality in the cecal ligation and puncture (CLP) sepsis model (13). Thus it has recently been concluded by general agreement that LPS injection may be an adequate model of endotoxic shock but not of sepsis (26). Because of these limitations, experimental models other than LPS injection have been developed to more precisely mimic the human disease process. One such model is the CLP model, which appears to closely mimic both qualitatively and quantitatively the clinical observations of vascular reactivity and inflammation induced during polymicrobial peritonitis, bacteremia, and systemic sepsis (9, 35).

Although there have been a number of studies that have focused on characterizing the hepatic, pulmonary, and systemic inflammatory responses that contribute to the pathogenesis of sepsis in the CLP model (26), unfortunately, only one study could be identified that investigated the effects of CLP on intestinal motility (18), and no molecular studies have been performed on the intestinal muscularis to our knowledge. The one motility study used rat ileal longitudinal muscle, which is now known to be relatively unsensitive to LPS products (6, 7, 34), and a nonselective nitric oxide inhibitor that also blocks constitutive enteric neural nitric oxide as well as all other nitric oxide synthases (18). Thus little functional and molecular data concerning polymicrobial sepsis leading to bowel dysmotility and gut-derived cytokine production exist. The experiments on the small and large intestine in the present study were designed to gain mechanistic insight into the molecular and functional motility consequences of polymicrobial sepsis in the clinically relevant polymicrobial cecal ligation and puncture sepsis model.

	MATERIALS AND METHODS

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Experimental groups and operative procedure. Sprague-Dawley male rats were obtained from Harlan (Indianapolis, IN). The University of Pittsburgh Institutional Animal Care and Use Committee approved the protocol. Rats were housed in a pathogen-free facility that is accredited by the American Association for Accreditation of Laboratory Animal Care and complies with the requirements of humane animal care as stipulated by the US Department of Agriculture and the Department of Health and Human Services. The rats were maintained on a 12:12-h light-dark cycle and provided with commercially available rat chow and tap water ad libitum.

Rats were anesthetized with continuous isoflurane inhalation. The operative procedures were performed under sterile conditions. Four groups of animals were studied (control, laparotomy, sham CLP, and CLP). CLP animals underwent a small midline laparotomy; the cecum was eventrated and partially ligated with a 4-0 silk tie and punctured once with a 21-gauge needle. CLP was performed to induce peritonitis in Sprague-Dawley rats weighing 230 g each. After induction of peritonitis, the rats were killed and studied at different time points (postoperative day 1, 2, 3, 4; n = 6 for each group) for functional studies and at different times (1, 3, 6, 24, 48 h; n = 6 per group) postoperatively for molecular studies. Age-matched rats not receiving an operation served as controls. Sham groups underwent laparotomy and cecal ligation without puncture or laparotomy with only minor cecal handling without either ligation or puncture.

Functional studies. Operated animals were anesthetized with isoflurane a second time at the time of death, and the abdominal wound was reopened. The entire bowel was then removed and placed in cold preoxygenated Krebs-Ringer buffer (KRB). Mechanical activity was measured as previously described (14). A segment of midjejunum or colon was pinned in a dissecting dish containing iced, preoxygenated KRB; the segment was opened along the mesentery. The mucosa was removed, and the remaining muscularis was cut into strips (1 x 10 mm) parallel to the circular muscle layer and suspended in standard horizontal mechanical organ chambers that were continuously perfused with preoxygenated KRB solution maintained at 37°C. One end of each strip was tied to a fixed post, and the other was attached to an isometric force transducer (WPI, Sarasota, FL). In the organ chamber, each strip was allowed to equilibrate for 1 h. Strips were then incrementally stretched to the length at which maximal contraction occurs. Dose-response curves were generated by exposing the muscles to increasing concentrations of the muscarinic agonist bethanechol (0.3–300 µM) for 10 min with intervening 10-min wash periods. The functional significance of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 inhibition was determined by measuring changes of in vitro circular muscle spontaneous contractility in response to selective iNOS inhibition using L-N⁶-(1-iminoethyl)-lysine dihydrochloride (L-NIL; 30 µM) and the COX-2 inhibitor 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl2(5H)-furanone (DFU; 5 mg/l). Contractile activity was calculated as grams per square millimeters per second by converting weight and length of the strip to square millimeters of tissue.

Intestinal transit was measured in control and operated animals at different times postoperatively by evaluating the intestinal location of nonabsorbable fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight = 70,000) as previously described (14). Animals were anesthetized and given FITC-dextran (200 µl of 5 mg/ml stock solution). Two hours after administration, the contents of the entire gastrointestinal tract were divided into the stomach, 10 equal small intestinal segments, cecum, proximal colon, middle colon, and distal colon. Each segment was opened and mixed vigorously with 2 ml of KRB solution to obtain a supernatant containing the FITC-dextran. The supernatant was centrifuged at 10,000 rpm to force the intestinal chyme to a pellet. The fluorescence in duplicate aliquots of the cleared supernatant was read in a multiwell fluorescence plate reader (GeminiXS, Molecular Devices, Sunnyvale CA; excitation 488 nm and emission 525 nm) for quantification of the fluorescent signal in each bowel segment. A median distribution histogram of the fluorescence contained in each bowel segment was then plotted for analysis of transit. This provided an accurate, nonradioactive measurement of intestinal transit without further surgical intervention.

To demonstrate the in vivo role of proinflammatory mediators, we performed in vivo gastrointestinal transit experiments with the specific iNOS inhibitor L-NIL and the COX-2 inhibitor DFU. We performed the CLP operation as described above. After 21 h, we injected L-NIL (50 µg/kg) and DFU (5 mg/kg) intraperitoneally and performed the transit procedure at 24 h as described above (n = 5).

Distal colonic in vivo motility was recorded in awake, restrained rats 24 h after colonic manipulation, sham operations, and controls (n = 5 each) with an intracolonic balloon-tipped catheter as previously described (25). Pressures were recorded and analyzed using the MacLab data acquisition package (ADInstruments, Castle Hill, Australia) and a Macintosh G3 computer. After a 20- to 30-min period of acclimatization, animals underwent recording for periods of 2–3 h.

Histochemistry and immunohistochemistry. Muscularis whole mounts were prepared as described earlier (16). Midjejunal and midcolonic segments were cut from the bowel and immersed in KRB in a Sylgard-filled glass dish at 4°C. The length and width of each jejunal segment were measured with a caliper, and the segment was gently pinned along the mesenteric border. The bowel was opened along the mesentery and stretched to 150% of the length and 250% of the width. Mucosa and submucosa were stripped off under microscopic observation (Wild, Heerbrugg, Switzerland), and the normalized, 5-cm opened segments of jejunum were fixed in 100% ethanol for 10 min. Each segment was washed twice in KRB, and the mucosa-free muscularis whole mounts were finally cut into 1 x 1 cm pieces and used for staining procedures.

Histochemical staining for myeloperoxidase (MPO) was used to detect polymorphonuclear neutrophils, which had infiltrated into the freshly prepared muscularis whole mounts using a mixture of 10 mg of Hanker-Yates reagent (Polysciences, Warrington, PA), 10 ml of KRB, and 100 µl of 3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO) for 10 min. The reaction was stopped with cold KRB. MPO-positive cells were counted at a magnification of x200.

Muscularis whole mounts were also used for immunohistochemical analysis of the jejunal and colon muscularis. Each whole mount was incubated for 24 h at 4°C in the primary antibody (ED1 and ED2) followed by three 10-min washes in buffer. The specimens were then incubated in the appropriate secondary antibody at 4°C for 4 h and washed three times for 10 min in buffer. Whole mounts were placed on a coverslip and inspected by fluorescent microscopy (Nikon FXA, Fryer, Huntley, IL).

Quantification of gene expression. The time course of postoperative mediator mRNA expression was analyzed using SYBRgreen two-step real-time RT-PCR (PE Applied Biosystems, Foster City, CA). Total mRNA extraction was performed as previously described using the guanidinium-thiocyanate phenol-chloroform extraction method(6). The RNA pellets were resuspended in RNAsecure resuspension solution (Ambion, Austin, TX). After the resuspension, a DNase treatment was carried out (DNA-free reagent, Ambion). Then, equal aliquots (20 µg) of total RNA from each sample, quantified by spectrophotometry, were processed for complementary DNA (cDNA) synthesis.

Primers were designed according to published sequences (17, 31, 36, 37) or using Primer Express software (PE Applied Biosystems) and purchased from Life Technologies (Rockville, MD). GAPDH was used as an endogenous control. The sequences of the real-time PCR primers are listed in Table 1. The efficiency and equality of the real-time PCR primer pairs were determined by amplifying serial dilutions of colonic muscularis cDNA. For each target gene, different MgCl₂ (2–5 mM) concentrations were tested to optimize the PCR amplification. Agarose gel electrophoretic analysis was used to verify the presence of a single product and to ensure that the amplified product corresponded to the size predicted for the amplicon. Each sample was estimated in triplicate. The PCR reaction mixture was prepared using the SYBRgreen PCR Core Reagents (Applied Biosystems). PCR conditions on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) were as recommended by the manufacturer. Relative quantification was performed using the comparative cycle threshold (CT) method as described previously by Schmittgen et al. (27) (see also User Bulletin no. 2, PE Applied Biosystems).

	RESULTS

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Gastrointestinal motility. We first sought to characterize functional alterations in gastrointestinal motility in response to nonlethal CLP-induced enteric polymicrobial bacteremia because this has not been previously accomplished (4, 35). Gastrointestinal transit was measured using orally administered FITC-labeled dextran after a period of 2 h in control animals, sham operated animals, and CLP animals on PODs 1–4 (Fig. 1).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Transit histogram for the distribution of nonabsorbable FITC-labeled dextran along the gastrointestinal tract 2 h after oral ingestion in control, sham, and cecal ligation and puncture (CLP)-treated animals. A: in control animals, the nonabsorbable marker accumulated in the last 3 segments of the intestine [geometric center (GC) = 9.3]. Sham animals had a similar distribution pattern (GC = 8.9; P = 0.365). CLP caused a significant delay in intestinal transit (GC = 4.9; P = 0.0032). B: plot of the calculated geometric center value for control, sham 24 h, and CLP at postoperative days 1–4 (CLP-1 to -4, respectively). The plot demonstrates the in vivo functional temporal recovery of gastrointestinal transit over a period of 4 days after the initial CLP-induced transit delay.

The above transit distribution histograms after 2 h primarily measured the functional movement of FITC-dextran through the stomach and small intestine. Therefore, to investigate in vivo colonic function after CLP, we performed intracolonic balloon pressure recordings in control, sham, and CLP animals after 24 h. Representative distal colonic pressure recordings from each group of animals are illustrated in Fig. 2.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Representative distal colonic pressure recordings obtained from a control (A), sham-operated (B), and CLP-manipulated (C) animal 24 h after surgery. CLP-induced sepsis caused a significant reduction in colonic contraction amplitude and frequency compared with control and sham-operated animals (P < 0.05).

In the next series of experiments, muscularis function was measured directly by determining the in vitro capacity of jejunal and colonic circular smooth muscle strips to respond to the cholinergic agonist bethanechol using standard organ bath techniques. As illustrated in Fig. 3, bethanechol exposure generated a dose-dependent increase in circular muscle contractile activity of the small bowel and colon.

View larger version (16K): [in this window] [in a new window]   Fig. 3. In vitro bethanechol dose-response curves of jejunal (A) and colonic (B) circular muscle strip contractility in control and CLP-treated animals. Contractility was measured and normalized to g·s–1·mm2 at increasing bethanechol concentrations in preoxygenated Krebs-Ringer buffer (KRB) at 37°C.

Leukocytic infiltration. Our previous studies have demonstrated that deterioration in muscle function after insult is frequently caused by the presence of leukocytes within the gastrointestinal muscularis. Therefore, leukocytic infiltrates within the small and large intestinal muscularis were investigated in whole-mount preparations 24 h after the induction of CLP-induced sepsis. We focused on macrophages, monocytes, and polymorphonuclear neutrophils because these leukocytes possess the ability to release smooth muscle kinetically active substances that directly or indirectly modulate the gut muscularis (7, 11, 15, 19, 29). As previously reported, ED2⁺ leukocytes (macrophages) form a dense network of resident phagocytes within the normal muscularis of the small and large intestines Fig. 4, A and B.

View larger version (53K): [in this window] [in a new window]   Fig. 4. Primary anitbody ED2 and ED1 immunohistochemistry of jejunal and colonic whole mounts showing resident macrophages and monocytes in control and CLP-manipulated animals. ED2-stained macrophages displayed a similar distribution pattern in jejunal (A) and colonic (B) whole mounts, showing a stellate appearance and multiple pseudopodia arising from the cytoplasm. As shown in the representative colonic whole mount 24 h after CLP-induced sepsis, macrophages changed their shape to a more rounded appearance with retracted pseudopodia (C). ED1 immunohistochemistry revealed rounded monocytes that were rarely seen in control jejunal sections (D), but the jejunal muscularis became significantly infiltrated after CLP (E) compared with controls. In contrast to the small intestine, the control colon displayed a large population of resident ED1+ monocytes, which could be seen to cluster around tightly adherent small vessels at the submucosal border of the colonic muscularis (F). As a result of this substantial resident monocytic presence in the colon, a distinction between control and CLP-manipulated (not shown) animals was not apparent. Original magnification: x200 for A–F.

As shown in Fig. 4D, jejunal whole mounts stained for the ED1 antigen revealed the presence of only a few round-shaped monocytes in control and sham animals. Additionally, as previously observed, this antibody also lightly stained the stellate-shaped resident muscularis macrophages. In the animals subjected to CLP, however, numerous ED1⁺ monocytes could be microscopically detected as newly extravasated leukocytes into the jejunal muscularis syncytium (Fig. 4E).

In contrast to the control jejunum, colonic muscularis whole mounts displayed a large number of constitutively present ED1⁺ cells, as well as the lightly stained ED1⁺ muscularis macrophages (Fig. 4F). These cells clustered heavily around tightly adherent small vascular structures that coursed along the submucosal edge of the muscularis externa. In fact, the irregular distribution and large basal presence of the round ED1⁺ cells made it difficult to quantitatively count an increase in ED1⁺ cells in colonic whole mounts prepared from CLP animals.

As shown in Fig. 5 and quantified in the histogram of Fig. 6, MPO-positive neutrophils were present only occasionally in control whole mounts of the jejunum and colon (16). Although the sham procedure of cecal eventration increased the number of extravasated PMNs in the muscularis of both organs, this polymorphonuclear neutrophils infiltrate was even more significantly enhanced in CLP animals.

View larger version (103K): [in this window] [in a new window]   Fig. 5. Myeloperoxidase (MPO) staining of jejunal and colonic whole mounts showing polymorphonuclear neutrophils (PMNs) after CLP compared with sham-operated and control animals. MPO-positive cells occurred only occasionally in control jejunal sections (A), and a slight increase was observed in sham-operated animals (B). CLP resulted in a significant recruitment of PMNs into the jejunal muscularis externa (C). Colonic whole mounts displayed a similar pattern of MPO-positive cells with few PMNs present in control (D) and a slight increase 24 h after sham-CLP operation (E). As shown in F, CLP caused a significant recruitment of PMNs into the colonic muscularis externa.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Histogram of MPO-positive cells quantifying the significant increase of infiltrating PMNs into both the jejunal and colonic muscularis after CLP compared with sham-operated animals and control whole mounts. Leukocytes were counted in 5 random fields at a magnification of x200 24 h after manipulation. Data are expressed as means ± SE; n = 6.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Real-time RT-PCR analysis of proinflammatory mediator mRNAs. Cycle threshold (CT) values were calculated in control, sham, and 1, 3, 6, 24, and 48 h after CLP-induced sepsis in the small bowel and colon. A: in the jejunum, IL-6 peaked after 3 h, whereas in the colon the peak was at 6 h. The fold increase showed regional differences, and was 3 times higher in the small bowel. B: IL-1 expressions in the small bowel and colon showed a similar temporal upregulation after CLP and a similar fold increase in the small bowel and the colon with a first peak at 3 h and a possible second peak at 48 h. C: monocyte chemoattractant protein (MCP)-1 peaked at 6 h in the small bowel and at 3 h in the colon after CLP. The small bowel had a second peak at 48 h. Fold increases were similar in both intestinal regions.

View larger version (32K): [in this window] [in a new window]   Fig. 8. Real-time RT-PCR analysis of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 mRNAs. CT values were calculated in control, sham, and 1, 3, 6, 24, and 48 h after CLP-induced sepsis in the small bowel and colon. A: in the jejunum, iNOS demonstrated a significant but relatively small increase, peaking at 6 h and declining back toward normal levels at 48 h. Colonic muscularis iNOS mRNA expression pattern was different in the colon with its maximum expression at 48 h, which exceeded the relative maximum increase in the small bowel by 25-fold. B: COX-2 levels demonstrated only minor induction after CLP-induced sepsis in the small bowel or the colon. In the colon, COX-2 mRNA expression had a tendency to decline during the first 6 h but rebounded to control levels again at 24 h, whereas in the small bowel, COX-2 had 2 moderate peaks at 1 and 48 h with a 3-fold increase compared with controls.

The functional contractile significance of the induction of iNOS and COX-2 mRNAs within the jejunal and colonic intestinal muscularis by CLP was investigated using the relatively selective iNOS inhibitor L-Nil (30 µM) and the COX-2 inhibitor DFU (5 mg/l). Circular jejunal and midcolonic muscle strips were harvested and prepared for organ bath recordings. After the equilibrated spontaneous contractions were recorded for a period of 30 min, muscles were acutely superfused with both L-Nil and DFU for a further 30-min period. Application of the iNOS and COX-2 blockers caused a 62 ± 10.0% increase over jejunal circular muscle control activity and an 181 ± 24.3% increase in colonic circular muscle spontaneous contractions over control colonic muscles exposed to the blockers.

	DISCUSSION

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Unfortunately, although many agents have shown promise in septic animal models, clinical trials of these treatments failed, and patient mortality rate with severe sepsis and subsequent multiorgan failure remains >20%. One critical reason for this discrepancy could be the use of clinically inappropriate animal models that do not closely mimic the septic human patient (4). Comparatively, CLP appears to be a reliable and clinically relevant animal model of the human septic condition. Indications for the relevance of the CLP model are that the endogenous polymicrobial nature of the induced infection is similar to clinical bacteremia and symptomatically after CLP animals develop an initial hyperdynamic cardiovascular response similar to what is observed in human sepsis (9, 26, 35). In this nonlethal CLP study, we observed the development of an intra-abdominal abscess surrounding the ligated and perforated cecum. The morphological appearance of perforation coverage and abscess formation mimics the clinical situation of a secondary intra-abdominal sepsis, such as after diverticulitis or gastric ulcer perforation with omental coverage (8). Like in our laboratory's previous studies using LPS-induced ileus (6, 7, 28, 33), we investigated the hypothesis that local inflammatory events within the gut muscularis externa and a subsequent inhibition of intestinal smooth muscle contractility mechanistically participate in the systemic inflammatory response syndrome and gastrointestinal ileus. Our aim was to investigate motility and cellular and molecular events within not only the small intestine, but also the colon, which has previously not been studied. Additionally, recovery from CLP-induced gastrointestinal ileus was also examined.

Functionally, we observed a substantial decrease in upper gastrointestinal transit after CLP, as indicated by the transit distribution histograms. Similarly, in vivo colonic pressure activity was suppressed, as well as in vitro jejunal and colonic muscle strip mechanical responses to bethanechol. The 50% degree in muscle inhibition was relatively similar between the jejunal and colonic regions of the gastrointestinal tract, as caused by our nonlethal CLP model. Interestingly, gastrointestinal transit started to recover after 24 h and reached a normal transit pattern again 4 days after the induction of sepsis. Thus, from a functional standpoint, nonlethal CLP suppressed circular muscle contractility to a similar extent and duration as did a single nonlethal injection of endotoxin (6, 7, 28). As reported in the one previous study using a lethal model of CLP (18), ileal longitudinal muscle decreased its function by 33%. The decreased sensitivity of longitudinal compared with circular muscle to CLP has also been observed with endotoxin (6, 34).

Muscle dysfunction in both the polymicrobial CLP and endotoxin models of sepsis is associated with a significant recruitment of neutrophils and monocytes into the jejunal muscularis 24 h after the initiation of sepsis (6, 7, 28). Quantitatively, however, neutrophil recruitment caused by CLP represented a greater percentage of the jejunal muscularis leukocytic infiltrate compared with the endotoxin model, which initiated a nearly pure monocytic recruitment into the jejunal muscularis (7). This more complex leukocytic infiltrate might be expected due to the intraperitoneal release of numerous bacterial products after polymicrobial CLP sepsis. The colonic muscularis was also significantly infiltrated with neutrophils during CLP. But unlike the small intestine, the control colonic muscularis contained a large population of resident ED1⁺ round-shaped monocytes, which clustered in a distinct perivascular pattern. A monocytic increase after CLP was difficult to ascertain in the colon, because of their large basal presence and irregular perivascular distribution.

We have previously demonstrated that MCP-1 is a critical chemokine involved in the recruitment of monocytes into the jejunal muscularis (33). Acute LPS injection causes a strong early 3-h 280-fold induction in jejunal MCP-1 as measured by real-time RT-PCR, whereas in the CLP model MCP-1 extracts peaked later at 6 h with only a 52-fold increase, as measured by real-time RT-PCR over control levels. Even a lower 30-fold peak increase over basal colonic MCP-1 express was measured after CLP. This lower colonic increase may partly explain the imperceptible change in colonic ED1⁺ monocytes after CLP but does not address the perivascular presence of monocytes in control tissues. Although we had the impression that basal colonic MCP-1 expression was higher compared with the small intestine based empirically on change in CT values, strict analysis of such a comparison between different tissue types is technically invalid.

It has been previously shown that stimulated resident and extravasated leukocytes within the muscularis externa generate an inflammatory milieu, which can perpetuate leukocyte recruitment and intestinal muscle dysfunction, which could lead to bacterial overgrowth and the further release of bacterial products (7, 12, 33). If this insidious cycle is not interrupted, systemic inflammatory response syndrome will develop, and thus the intestine can easily be appreciated as the "motor" of multiple organ failure (20, 24). In this study, we show the development of such a cascade of inflammatory events in response to polymicrobial sepsis. Real-time RT-PCR analysis of jejunal and colonic muscularis extracts demonstrated a significant upregulation in the prototypical proinflammatory cytokines IL-6 and IL-1, as well as the inflammatory synthases iNOS and COX-2. It has been shown previously that iNOS and COX-2 can be induced by a variety of bacterial products, cytokines, hormones, and growth factors during inflammation. In this study, CLP caused a significant increase in iNOS mRNA in small intestinal and colonic muscularis extracts. Interestingly, however, the timing and degree of the induction appeared quite organ specific. Small intestinal extracts demonstrated peak mRNA induction at 6 h, whereas the colonic signal peaked much later at 48 h. It is difficult to explain this temporal difference in iNOS induction, but it could be because the colon is more anatomically insulated from the small intestine and, therefore, less rapidly exposed to the bacterial leakage from the punctured cecum. However, a region-specific heterogeneous response of the constituents of the different muscularis extracts cannot be ruled out. Here we demonstrated for the first time that a large population of ED1⁺ monocytes with a perivascular distribution is constitutively present in the colon. The presence of these phagocytes, in addition to the ED2⁺ resident muscularis macrophages could explain the higher degree of iNOS induction within colonic extracts compared with the small intestine, which does not display this pattern of resident monocytes. We have also observed this regional difference in iNOS expression after surgical manipulation of the intestine, which our laboratory used to investigate the mechanisms of postoperative ileus (21, 32). However, in contrast to iNOS, COX-2 was more highly induced in the small intestine compared with the colon, although the regional difference was not as striking. The molecular induction of iNOS and COX-2 was functionally correlated with jejunal and colonic contractile responses after synthase blockade with L-Nil and DFU. Spontaneous circular muscle contractile activity recorded from both the jejunum and colon harvested from CLP animals increased significantly over control muscle responses to synthase blockade. Additionally, simultaneous in vivo administration of iNOS and COX-2 inhibitors almost completely restored the transit after CLP, supporting the in vitro data of L-NIL and DFU usage followed by an improved muscle activity outcome. Also reflected in contractile activity was the observation that colonic activity increased to a greater percentage of control activity compared with the jejunum, thus demonstrating that both regions of the gastrointestinal tract were tonically inhibited by the upregulation of iNOS and COX-2 synthases and also that functional activity correlated with a greater iNOS induction within the colonic muscularis.

In summary, the above data demonstrates that polymicrobial CLP sepsis induces a complex inflammatory response within the intestinal muscularis with the recruitment of leukocytes and elaboration of mediators that inhibit intestinal muscle function. Interestingly, differences were elucidated between LPS and CLP models of sepsis as well as a heterogeneous regional response of the gastrointestinal tract to CLP. Thus the intestine is not only a source of bacteremia but also an important target of the bacterial products with major functional consequences to intestinal motility and the generation of cytokines, which could participate in the development of multiple organ failure.

	GRANTS

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This work was supported by National Institute of of General Medical Sciences Grants R01-GM-58241 and P50-GM-53789.

	ACKNOWLEDGMENTS

 
The authors thank Jessica Whitcomb for excellent technical support regarding the in vivo transit and immunohistochemistry experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. J. Bauer, Dept. of Medicine/Gastroenterology, Univ. of Pittsburgh Medical School, S849 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: tbauer{at}pitt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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