Division of Gastroenterology, Departments of Surgery and Medicine, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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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-N6-(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.
sepsis; inducible nitric oxide; small intestine; macrophage; smooth muscle; lipopolysaccharide
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INTRODUCTION |
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GASTROINTESTINAL (GI) dysmotility frequently occurs during sepsis and multisystem organ failure, yet the cellular mechanisms involved in this process remain an enigma. Current evidence supports the hypothesis that bacterial toxins, particularly endotoxin, and cytokines generated primarily by the gut, act as causative factors of sepsis-induced ileus (4). Lipopolysaccharide (LPS) is the prototypic activator of the macrophage, and embedded within the intestinal muscularis externa resides an impressive network of resident macrophages (15). We have previously shown that the normally quiescent network of intestinal muscularis macrophages is rapidly activated during endotoxemia (5). Endotoxemia also initiates a moderate leukocytic infiltrate within the muscularis and causes a profound reduction in rat intestinal circular smooth muscle mechanical activity (5). Activated macrophages are remarkable protean cells that secrete in excess of 100 different substances, including prostaglandins, leukotrienes, cytokines, and nitric oxide (NO) (1, 18, 32). Many of these factors are known to directly alter the kinetic properties of GI smooth muscle and could be potential mediators of intestinal ileus.
NO is produced from the amino acid L-arginine by the enzyme NO synthase (NOS). Three isoforms of NOS have been described in the literature (3, 20). Two of these isoenzymes are found in neuronal and endothelial tissues and appear to be constitutively expressed, requiring calcium and calmodulin as cofactors. Neuronal NOS has been shown to be extensively distributed throughout the GI tract within the enteric nervous system (19), and NO is known to be the major inhibitory neurotransmitter in the human small intestine (25). Endothelial NOS is found throughout the endothelium and regulates vascular tone. The third isoform, inducible NOS (iNOS) is found in a wide range of cell types and its activity is independent of increases in intracellular calcium (3, 33). The induction of iNOS by LPS results in NO levels that are several orders of magnitude greater than that generated by the constitutive NOS isoforms (20, 33). All three isoforms of NOS are competitively inhibited by L-arginine analogs such as NG-nitro-L-arginine (L-NNA). In contrast, L-N6-(1-iminoethyl)lysine (L-NIL) is a 28-fold more selective inhibitor for iNOS (16). These agents have been useful in defining some of the specific biological effects attributed to a particular NOS isoform (21).
We hypothesize that NO, specifically from the intestinal muscularis macrophage network, is an important mediator of LPS-induced gut dysmotility. Therefore, the objective of this study was to determine the role of induced NOS in the modulation of rodent intestinal motility during endotoxemia.
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METHODS |
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Animals. Male ACI (Black agouti) rats weighing 180-240 g were obtained from Harlan Sprague-Dawley (Indianapolis, IN), male C57BL/6 mice weighing 18-20 g were obtained from Charles Rivers Laboratories (Wilmington, MA), and male iNOS knockout mice, confirmed by Southern blot analysis, were obtained from Merck Research Laboratories (Rahway, NJ). All animals were kept in accordance with the University of Pittsburgh Animal Care and Use Committee guidelines, housed in a room with a 12:12-h light-dark cycle, and fed commercially available rat chow and tap water ad libitum. The experimental design was approved by the University of Pittsburgh Animal Care and Use Committee.
Endotoxemia was induced in rats and mice by a single intraperitoneal injection of LPS (Escherichia coli 0111:B4) obtained from Sigma Chemical (St. Louis, MO) at 15 mg/kg. Rats were killed at various time points (1, 3, 6, 12, and 24 h), and functional studies were performed. Mice were all killed 12 h after injection.iNOS mRNA studies.
The rat jejunal muscularis was isolated from the mucosa-submucosa by
slipping 5-cm-length portions of the intestine over a glass rod and
stripping the muscularis from the jejunal mucosa. The isolated jejunal
muscularis was snap frozen in liquid nitrogen and stored at
70°C. Total RNA extraction was performed as previously described (8) using the guanidinium thiocyanate phenol-chloroform extraction method. Briefly, extracted RNA concentrations were determined using a spectrophotometer, equal aliquots of total RNA from
each sample were used for cDNA synthesis, and amplification of
synthesized cDNA was carried out using polymerase chain reaction with
32P-labeled primers. The primer
pairs were targeted to known sequences of iNOS that were designed as
described by Innis and Gelfand (8). The specificity of the individual
PCR primers was confirmed by sizing the PCR products and by subjecting
the PCR products to analysis by restriction enzyme digestion (28).
Target regions to be amplified within each different cDNA were chosen
to facilitate this confirmation of specificity and to allow
differentiation of end products resulting from amplification of
contaminating genomic DNA. Sequences of the PCR primers were as
follows: iNOS 5'-TTGGGTCTTGTTAGCCTAGTC and
3'-TGTGCAGTCCCAGTGAGGAAC (accession number D12520; product size
264 bp).
Muscularis dissociation and primary culture. The cellular constituents of the intestinal muscularis were dispersed and isolated in culture as previously described (11). Briefly, the entire small intestine of the anesthetized rat was flushed with 10 ml of cold Ringer solution and resected, leaving the colon in situ. The small intestine was transferred to a sterilized beaker with Hanks' balanced salt solution (HBSS) from Sigma Chemical containing 100 U/ml penicillin G and 100 µg/ml streptomycin. The gut was cut into 5-cm lengths, and each segment was pinned down to the Sylgard bottom of a glass dish. After resection of extraneous mesenteric tissues, the segment was slipped onto a glass rod. The muscularis was gently incised along the mesenteric border and circumferentially stripped from the submucosa by the use of a moist cotton-tip applicator. The stripped muscularis was collected in a beaker containing iced magnesium- and calcium-free HBSS. After collection, the isolated muscularis was put into an enzymatic digestion mixture containing 0.6 mg/ml collagenase D, 0.1 mg/ml DNase I, and 2.0 U/ml dispase II from Boehringer Mannheim (Indianapolis, IN) diluted in magnesium- and calcium-free HBSS. The tissue was incubated in a rotary shaker at 37°C for 20 min and centrifuged at 500 g for 5 min, and then the supernatant was discarded and the pellet resuspended with 10 ml HBSS. After an additional incubation in the shaker for 20 min the test tube was vigorously shaken to augment dispersion. The cellular suspension was filtered through a nylon mesh, centrifuged at 500 g for 5 min at 4°C, and washed with HBSS three times at 400 g for 5 min at 4°C. The resulting pellet was resuspended in RPMI, and cell viability was assessed with trypan blue. Cells were counted and cultured in 75 cm2 flasks at a density of 3 × 105 cells/flask. On reaching confluence, cells were detached and dissociated using a cell dissociation solution with 0.125% trypsin. Detachment was monitored under a phase-contrast microscope, and the reaction was stopped with supplemented medium and a centrifugation wash cycle. The isolated cells were then dispersed with trituration and reseeded in new flasks at a density of 1 × 105 cells/ml. The passaged cellular constituents of the entire muscularis were fed by changing the medium every 2 days.
Immunohistochemistry.
Immunohistochemistry was performed on whole mounts of rat jejunal
muscularis. Whole mounts were prepared from segments of the midjejunum
after fixation with ethanol (100%) for 10 min (5, 11). Fixed whole
mounts were stained with a primary antibody followed by the appropriate
fluorescent secondary antibody. Specimens were mounted with Gel-Mount
obtained from Biomeda (Foster City, CA) and inspected by fluorescent
microscopy. The following primary and secondary antibodies were used:
iNOS 1:200 monoclonal antibody (MAb) mouse, ED2 1:100 MAb mouse,
-actin 1:200 MAb mouse, and goat anti-mouse IgG 1:50 secondary to
mouse antibodies.
Functional studies. All animals were anesthetized by inhaled methoxyflurane. A segment of intestine from the midjejunum of each animal was removed through a midline incision and submerged in an iced, preoxygenated Krebs-Ringer solution (KRB). Circular muscularis strips were prepared as previously described by Eskandari et al. (5). Contractile force was assessed using standard organ bath techniques. Briefly, one end of each strip was attached to an isometric force transducer by 4.0-silk sutures and the other end was pinned down in the organ bath. Each bath was continuously perfused with a preoxygenated, preheated (37°C) KRB that was continuously monitored with a thermistor probe. Muscles were allowed to equilibrate in the chamber for 30 min and then stretched in a step-wise fashion to optimal length. Spontaneous contractile activity was measured in the presence and absence of L-NIL at a concentration of 30 µM or L-NNA at 100 µM, both of which were obtained from Sigma Chemical. Contractile activity was normalized to grams per square millimeter per second for all strips. Spontaneous and bethanechol-stimulated contractions were recorded, measured, and stored in a computer using A/D hardware and the Acknowledge software package from Biopac Systems (Santa Barbara, CA).
Electrical field stimulation. Isolated circular muscularis strips were prepared and placed in a continuously perfused organ bath chamber. The bath was continuously perfused with preoxygenated, preheated (37°C) KRB, and each muscle was allowed to equilibrate for a period of 30 min, a minimum period of time to minimize the in vitro expression of iNOS. TTX-sensitive neural contractile responses were elicited by electrical field stimulation (EFS) with two platinum wires running parallel alongside the muscle strip (pulse duration 0.8 ms, frequencies ranging from 1 to 20 Hz, stimulus durations ranging from 1 to 100 s, voltage 150 V). Square-wave voltage pulses across the platinum wires were generated by a S11 voltage stimulator connected to a SIU-5 stimulus isolation unit from Grass Instruments (Quincy, MA).
Data analysis. Results are expressed as means ± SE. Data were statistically analyzed using an unpaired Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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iNOS mRNA.
LPS has been demonstrated to be the prototypic activator of macrophages
and a potent stimulus for the induction of iNOS (20, 33). To determine
if iNOS expression was upregulated in the intestinal muscularis during
endotoxemia, iNOS mRNA levels in the isolated muscularis was measured
by semiquantitative RT-PCR in controls and LPS-injected animals.
RT-PCR, using a rat-specific iNOS primer, revealed low, but detectable
levels of iNOS expression in the muscularis of control animals as seen
in Fig. 1. Pretreatment with a single bolus
injection of LPS (15 mg/kg) caused a rapid and significant increase in
rat muscularis iNOS mRNA levels, peaking at 3 h and returning to
baseline at 12 h. As illustrated in the histogram of Fig. 1, iNOS mRNA
expression in LPS-treated rats was 20-fold greater at the 3-h time
point compared with controls (n = 4, P < 0.05). Anesthetized
saline-injected animals also served as controls and did not differ from
uninjected control animals (data not shown).
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iNOS immunohistochemistry.
We have observed the normal presence of a dense network of resident
macrophages within the jejunal muscularis externa of the rat (Fig.
2A),
which possess the known LPS receptor CD14. To localize the cell types
expressing the iNOS protein, immunohistochemistry was performed using a
specific iNOS MAb. As illustrated in Fig. 2C, 12 h after LPS pretreatment, the
dendritic-appearing, ED2-positive muscularis macrophages became
positively labeled. Muscularis whole mounts prepared at 6 and 24 h after LPS injection also demonstrated iNOS immunoreactivity,
but most prominent staining was observed in the 12-h specimens. No
iNOS-positive cells were detected in the muscularis of control rats
(Fig. 2B).
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iNOS blockade.
Using standard organ bath techniques, we found that a single
intraperitoneal bolus injection of LPS (15 mg/kg) caused a significant reduction in rat spontaneous circular muscle contractility at 12 h.
Control spontaneous circular muscle generated 0.41 ± 0.062 g · mm2 · s
1,
whereas LPS pretreatment (15 mg/kg) caused a 68% reduction in contractile area to 0.13 ± 0.026 g · mm
2 · s
1
(P < 0.05, n = 10). The acute in vitro
application of the nonspecific NOS inhibitor
L-NNA (100 µM) caused a 85%
increase in circular smooth muscle spontaneous activity in LPS-treated
rats, but only a 21% increase in control muscle activity (normalized
to
g · mm
2 · s
1,
n = 5, P < 0.05; Fig.
4). To investigate the specific role of induced NOS in the suppression of contractile activity, we exposed circular smooth muscle strips to the specific iNOS inhibitor
L-NIL (30 µM) (16). As shown
in Figs. 4 and 5,
L-NIL caused a significant (53%) reversal in the impaired circular smooth muscle activity of
LPS-treated rats, whereas control muscle exhibited no response to
L-NIL
(n = 5, P < 0.05).
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iNOS knockout mice.
To confirm the role of inducible NO as a possible mechanism mediating
LPS-induced smooth muscle suppression, animals genetically deficient
for iNOS gene (knockout mice) were studied (13). As seen in the rat
experiments, L-NIL (30 µM) did
not significantly increase spontaneous contractile activity recorded
from the mouse jejunal muscle strips of untreated control (C57BL/6) or
untreated iNOS knockout mice. Also, similar to rat experiments,
circular muscle mechanical activity recorded from the jejunum of LPS
(15 mg/kg, n = 4)-pretreated wild-type
mice demonstrated a significant 57% suppression in spontaneous muscle
activity. When the LPS-pretreated wild-type muscles were exposed to
L-NIL, a significant increase in
spontaneous muscle strip activity (44%) was recorded, thus demonstrating an iNOS-dependent reversal of the impairment of circular
smooth muscle activity (n = 4, P < 0.05). When iNOS
knockout animals were pretreated with LPS, spontaneous activity was
depressed significantly less than in wild types (38%), and
L-NIL did not change spontaneous
muscle contractile activity (Fig.
6).
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DISCUSSION |
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This study demonstrates that NO from iNOS plays a central role in mediating LPS-induced suppression of intestinal smooth muscle activity. Although neural NO is known to be a potent inhibitory neuromuscular transmitter in the GI tract (25), the specific pathophysiological effect of inducible NO on gut motility has not yet been elucidated. We observed that a single intraperitoneal bolus injection of LPS caused an increased expression of iNOS mRNA within the isolated muscularis of the intestine. The majority of iNOS protein appeared to be localized to the dense network of muscularis macrophages that normally reside within the muscularis externa of the bowel wall. LPS injection and the subsequent upregulation of iNOS were followed by a marked decrease in spontaneous circular smooth muscle contractility activity. The selective iNOS inhibitor L-NIL significantly reversed the LPS-induced impairment of circular smooth muscle activity. The role of iNOS in mediating LPS-induced contractility changes was further established using iNOS knockout mice. These genetically deficient animals did not exhibit the L-NIL-sensitive increase in circular smooth muscle activity after LPS treatment, which was observed in both the rat and control B6 mice. Taken together, these observations implicate the role of inducible NO generated from the muscularis macrophage network in the pathogenesis of sepsis-induced intestinal ileus.
We have previously shown that the extensive network of normally quiescent macrophages within the intestinal muscularis is activated in response to LPS (5). The same study also demonstrated that LPS caused a mild proinflammatory response within the muscularis consisting of neutrophils, mast cells, and monocytes. Activated macrophages are remarkable protean cells, which secrete in excess of 100 different substances, including prostaglandins, leukotrienes, cytokines, and NO (1, 2, 17, 25). Many of these factors are known to directly alter the kinetic properties of GI smooth muscle (24) and could be potential mediators of intestinal ileus. In addition to these resident cells, LPS-recruited leukocytes are also known to produce substances that have known contractile altering effects (i.e., reactive oxygen intermediates) (6, 10).
The current study extends our previous observations by showing that the activation process results in the upregulation of iNOS mRNA within the isolated muscularis of the small intestine. This increase in iNOS mRNA was observed to occur before the onset of the cellular inflammatory response associated with the intraperitoneal injection of LPS. Also important is our observation that iNOS is apparently primarily localized to the dense network of macrophages that normally reside within the muscularis externa. We cannot exclude the induction of iNOS within the intestinal smooth muscle cells under these in vivo and in vitro conditions. However, it would appear that intestinal muscle cells are different from vascular smooth muscle cells, which have been reported to abundantly express iNOS during endotoxemia (3, 20).
A myriad of biological effects has been attributed to NO. In particular, NO has been shown to be the major bacteriocidal defense mechanism of macrophages. LPS, a causative agent of sepsis, is known to be a potent stimulus for the activation of macrophages and the induction of iNOS (27, 33). In this study, LPS caused a marked increase in iNOS mRNA expression within the isolated intestinal muscularis within only 3 h of endotoxemia, suggesting a primary response to LPS. The increased expression of iNOS mRNA could potentially be derived from a variety of cell types within the muscularis externa, including resident macrophages (5), endothelial cells, fibroblasts, glial cells, and infiltrating phagocytes (22). However, the induction of iNOS mRNA occurred before the onset of leukocyte recruitment into the muscularis in response to LPS. Through immunohistochemical techniques we were able to demonstrate iNOS protein expression localized to a dense network of dendritic, stellate-shaped cells having a similar morphology and pattern of distribution within the muscularis as the resident muscularis macrophages. The use of double-labeling techniques for macrophages (ED2) and iNOS in chambers slides of dispersed muscularis cells that were cultured in the presence of LPS confirmed this observation. Albeit, the presence of other iNOS-positive immunoreactive cellular populations were not clearly detected, we cannot completely rule out their contribution to our increased iNOS message levels.
Although NO is known to be an important inhibitory neuromuscular transmitter in the GI tract, only recently has inducible NO been suggested to play a role in gut dysfunction, particularly during endotoxemia (5, 30, 31). Recently, Eskandari et al. (5) showed that rat jejunal circular smooth muscle spontaneous and bethanechol-stimulated activity was suppressed by 68 and 91%, respectively, 24 h after a single bolus intraperitoneal injection of LPS. In contrast, LPS caused only a 20% reduction in longitudinal smooth muscle activity (5, 30). The greater sensitivity of the circular muscle demonstrated in our experiments correlates with the predominance of iNOS immunoreactive resident macrophages lying within the circular muscle layer. Wirthlin et al. (31) found that LPS caused an increase in both the constitutive and inducible isoforms of NOS within whole gut extracts and that nonselective NOS blockade reversed the effects of LPS-induced accelerated intestinal transit in the rat. The increase in NOS mRNAs in this study may have come from both mucosa-lamina propria cells (7) and the muscularis. Additionally, amelioration of accelerated transit in LPS-treated animals by the nonselective synthase blocker L-NNA was probably due to competitive inhibition of both constitutive and iNOSs. The same year Weisbrodt et al. (30) also reported that the nonselective NOS blocker L-NNA partially reversed the mild LPS-induced suppression of rat longitudinal smooth muscle activity. In this study, we attempted to delineate the specific contribution of iNOS to this process by using L-NIL, a selective iNOS inhibitors, and the nonselective NOS blocker L-NNA. The in vitro perfusion of L-NNA on control circular muscle strips caused a significant increase in spontaneous contractile activity, suggesting a competitive inhibition of neural NOS activity. In LPS-treated animals, L-NNA caused an even greater increase in spontaneous contractile amplitudes, which correlates with the induction of NOS by LPS and the subsequent antagonism by L-NNA. These data are consistent with the known inhibitory effects of NO on GI smooth muscle. In contrast to nonselective blockade, L-NIL did not significantly alter the contractility of control muscle strips but did improve in vitro circular smooth muscle spontaneous activity of LPS-treated animals. The selectivity of L-NIL for iNOS was further supported by the observation that EFS-induced neural relaxations were not inhibited by L-NIL at a similar concentration (30 µM). In this study the acute application of L-NIL (30 µM) did not completely reverse all the effects of LPS treatment. It must be pointed out that L-NIL is not a "completely" selective blocker at higher concentrations, but that the concentration used in this study (30 µM) had no effect on the neurally mediated relaxations. However, experimentally choosing this concentration was probably at the expense of an incomplete blockade in iNOS activity. An alternative hypothesis or additional mechanisms, which are plausible to explain the residual effects of LPS, is that excessive inducible NO may have caused nitrosylation of the contractile elements and/or that reactive nitrogen and oxygen intermediates caused damage to the circular smooth muscle cells. Furthermore, our data do not rule out that a component to the suppression in activity may be due to the induction of a constitutive form of NOS (31).
By using genetically engineered mice deficient for the iNOS gene (13), we were able to substantiate the role of iNOS in mediating LPS-induced circular smooth muscle suppression. In B6 control mice, L-NIL did not change spontaneous muscle activity, but it did increase the activity of muscle strips from LPS-treated mice. The 44% increase in activity is comparable to the 53% increase we observed in the rat. Additionally, L-NIL increased bethanechol-stimulated contractions recorded from LPS-treated wild-type mice. In contrast, L-NIL did not appreciably change the contractility of muscle strips from either LPS-treated or -untreated iNOS knockout mice as might be expected.
In conclusion, these experiments demonstrate a novel immuno-muscular interaction via induced NO, which significantly alters intestinal motility during endotoxemia. LPS not only primes and activates the dense muscularis macrophage network for the production of proinflammatory cytokines but also elicits a significant increase in iNOS protein expression by these resident leukocytes. The suppression of circular smooth muscle contractile activity by LPS seems to be at least partially mediated by this macrophage-derived NO. Therefore, NO produced by the network of muscularis macrophages appears to play a major role in the complex cascade of events leading to sepsis-induced ileus.
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ACKNOWLEDGEMENTS |
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This study was supported in part by Grants from the Deutsche Forschungsgemeinschaft Ka-1270/1-1, R01-GM-58241, and RO1-GM-44100, and the National Institutes of Health P50-GM-53789.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. J. Bauer, Dept. of Medicine/Gastroenterology, 572 Scaife Hall, 3550 Terrace St., Univ. of Pittsburgh Medical School, Pittsburgh, PA 15261 (E-mail: tbauer{at}pitt.edu).
Received 11 March 1999; accepted in final form 4 May 1999.
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