1Gastrointestinal and Neuroscience Research Groups, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada; 2Departments of Medical Physiology and Surgery, University Medical Centre, Utrecht, The Netherlands; and 3Department of Gastroenterology, University Medical Centre, Nijmegen, The Netherlands
Submitted 14 February 2003 ; accepted in final form 22 July 2003
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ABSTRACT |
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myenteric plexus; submucosal plexus; cyclooxygenase-2; vasoactive intestinal polypeptide; nitric oxide synthase
Although direct excitatory effects of inflammatory mediators have been observed in the majority of enteric neurons examined by electrophysiology, selective activation of subsets of enteric neurons has also been observed by morphological approaches and is based on the expression of the inducible gene product Fos. For example, incubation of guinea pig ileum with PGE2 caused a concentration-dependent increase in Fos immunoreactivity in submucosal neurons but not in myenteric neurons (10). Double-labeling immunohistochemistry revealed that Fos was localized exclusively in neurons that express vasoactive intestinal polypeptide (VIP), but not those that express neuropeptide Y (NPY). From this it was concluded that PGE2 selectively induces Fos in noncholinergic secretomotor/vasomotor neurons (which are chemically coded by VIP) of the guinea pig submucosal plexus. This approach has also been used to show activation of specific neuronal populations in other systems. Thus Schicho et al. (37) concluded that gastric acid challenge stimulates a subpopulation of nitrergic, but not cholinergic, myenteric plexus neurons in the stomach. Recent electrophysiological studies have revealed that in the guinea pig myenteric plexus, the cytokines interleukin (IL)-1 and IL-6 act on a much smaller population of neurons in the ileum than in the colon (18, 19).
Cytokines are polypeptide mediators produced by activated immune and epithelial cells in the inflamed intestine that influence the activity of other cells including neurons (5). The prototypic proinflammatory cytokine IL-1 is released by activated immune cells and is increased in the mucosa of patients with inflammatory bowel disease (7, 21) and in the inflamed intestine in animals (6). In isolated preparations, application of IL-1
has been shown to evoke an increase in excitability of submucosal and myenteric neurons, and also to mediate distinct effects on cholinergic and noradrenergic transmission (1, 5, 18, 19, 44). In the myenteric plexus of the ileum, only a small proportion of neurons were activated by IL-1
, whereas in the myenteric plexus of the colon and submucosal plexus of the ileum, IL-1
evoked a slow depolarization in the majority of neurons (18, 19). The reasons for these differences presumably lie in the expression of IL-1 receptors, although these have not yet been examined.
In the present study, we have used Fos expression to assess the effects of IL-1 in isolated preparations of guinea pig ileum and colon to address whether specific, chemically coded neuronal populations are activated by this cytokine. We tested the hypothesis that IL-1
activates all classes of enteric neuron. We used the expression of Fos as a marker of neuronal activation that also provides a way to assess activation of a population of neurons with single-cell resolution (39). We also examined if the effects of IL-1
were mediated through a PG-mediated pathway and whether IL-1
induced the expression of cyclooxygenase (COX)-2, the inducible enzyme responsible for PGE2 synthesis in inflammation.
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MATERIALS AND METHODS |
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In vitro stimulation with human recombinant IL-1. Animals were anesthetized with halothane (5-3% after induction of anesthesia in oxygen). A 20-cm segment of distal ileum 4 to 24 cm from the ileocecal junction and a 10-cm segment of distal colon 2 to 12 cm from the rectum were carefully removed and immersed in oxygenated ice-cold Krebs solution of the following composition (in mM): 117 NaCl, 4.8 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, and 11 glucose, pH 7.4, gassed with 95% O2-5% CO2. The solution also contained the L-type calcium channel antagonist nifidepine (1 µM; Sigma, St. Louis, MO) to prevent motility.
After a 5-min rest, the ileal segments were divided into four pieces of 5 cm and the colonic segments were divided into four pieces of
2.5 cm. Three segments each of colon and ileum were transferred to Krebs solution maintained at 37°C. The remaining one ileal segment and one colonic segment from each animal were immediately dissected along the mesenteric border, pinned flat in a petri dish lined with a silicone elastomer (Dow Corning, Midland, MI), and fixed overnight in Zamboni's fixative at 4°C and then processed for immunohistochemistry (see below). These segments served as absolute controls for the induction of Fos and COX-2. Test segments were then incubated (at 37°C) for a total time of 140 min: 20-min preincubation with normal Krebs solution or Krebs solution containing indomethacin (10 µM, Sigma), 60 min (2 x 30 min) in normal Krebs solution, Krebs solution with human recombinant IL-1
(hrIL-1
; R&D Systems, Minneapolis, MN), or Krebs solution with hrIL-1
and indomethacin, and 60 min (2 x 30 min) in which all tissues were maintained in normal Krebs solution. The tissues incubated in Krebs alone for 140 min served as incubation controls. After incubation, the tissues were rinsed in ice-cold PBS (pH 7.4), cut open along the mesenteric border, pinned, fixed overnight in Zamboni's fixative at 4°C, and processed for immunohistochemistry as described below.
Immunohistochemistry. Whole mount preparations of the myenteric and submucosal plexuses were processed for indirect immunohistochemistry as previously described (10, 25). In short, the preparations were pinned flat on a silicone-coated petri dish, and the mucosa was removed by gentle stroking with fine forceps. The myenteric plexus and submucosal plexus were separated by dissection from each other. The circular muscle was removed. The preparations were exposed for 36-48 h at 4°C to one or more primary antibodies. The primary antibodies used are described in Table 1. After incubation and washing in PBS (3 x 10 min), the tissues were then incubated in appropriate secondary antibodies for 1-2 h at room temperature. Secondary antibodies used were: donkey anti-rabbit-CY3 and anti-mouse CY3, donkey anti-rabbit FITC, rabbit anti-mouse FITC, and goat anti-rabbit FITC (1:100 each; Jackson ImmunoResearch Laboratories, West Grove, PA). After washing in PBS (3 x 10 min), tissues were mounted in bicarbonate-buffered glycerol (pH 8.4) and examined by using a Zeiss Axioplan fluorescence microscope. Photographs were taken by using a digital imaging system consisting of a digital camera (Sensys; Photo-metrics, Tuscon, AZ) and image analysis software (V for Windows; Digital Optics, Auckland, New Zealand).
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To assess the proportion of the immunoreactivity to anti-Fos (TF161), the neurons labeled with anti-Fos TF161 were compared with neurons labeled with the panneuronal marker Fos4 (Santa Cruz Biotechnology, SC-52, 1:1,000) (30). By using this method, we quantified Fos immunoreactivity as a percentage of total neurons. In whole mount preparations, five myenteric ganglia and 20 submucosal ganglia were counted in both ileal segments (n = 12) and colonic segments (n = 12). By labeling the COX-2-producing cells, one can draw a qualitative conclusion of the mechanism of the effect of the cytokine IL-1. Finally, by using S-100, we were able to distinguish enteric glia cells from enteric neurons within the ganglia, and the number of activated enteric glia could be assessed.
Statistical analysis. All experiments involved at least three separate trials. Values are expressed as mean ± SE. The Student's unpaired t-test was used to compare two means between two experiments, whereas a one-way ANOVA followed by a post hoc analysis by using a Bonferroni corrected t-test was used when comparing more than two means. Data were considered statistically significant when P < 0.05.
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RESULTS |
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After incubation with hrIL-1, Fos immunoreactivity was localized to both large and small nuclei in ganglia of the myenteric and submucosal plexuses. The large nuclei were generally brighter and had visible (unlabeled) nucleoli, consistent with them being the nuclei of enteric neurons. To identify these further, use was made of an antibody (referred to as neuronal nuclear antibody) that labels a constitutive nuclear antigen found only in enteric neurons (30). All large Fos immunoreactive nuclei were labeled with this antibody, which was also used to assess the proportion of total neurons in all subsequent data (Fig. 1). The small nuclei were generally fainter, did not have visible nucleoli, and in double-labeling studies were surrounded by a cytoplasm that was immunoreactive for the enteric glial marker S-100 (Fig. 1). In subsequent data, nuclei that were Fos positive/neuronal nuclear antibody negative were categorized as enteric glia.
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Fos expression is increased in neurons after incubation with hrIL-1. Incubation of ileal and colonic tissue with hrIL-1
in the lowest concentration tested (0.1 ng/ml) for 1 h resulted in a significant increase in Fos immunoreacting compared with untreated controls in the ileal and colonic myenteric plexus, but not in the submucosal plexus of either region of the bowel (Fig. 2). Increasing concentrations of hrIL-1
did not result in a concentration-dependent increase in Fos expression in the myenteric plexus of the ileum in which
10-13% of neurons were activated. In contrast, increasing concentrations of hrIL-1
caused a concentration-dependent increase in Fos expression in the colonic myenteric plexus with low concentrations activating
15% increasing to 42% of neurons at the highest concentration tested (Figs. 2 and 3).
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In the submucosal plexus, there was a concentration-dependent increase in Fos expression in both regions examined (Figs. 2 and 4). In the ileal submucosal plexus, 60% of neurons were activated at the highest concentration of hrIL-1
used, whereas in the colon this reached 75%.
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Fos expression in enteric neurons is attenuated in the presence of indomethacin. To examine whether the Fos expression was a direct action of hrIL-1, we incubated tissues in indomethacin before application of hrIL-1
. In both regions of the bowel and in both enteric plexuses, Fos expression was virtually abolished in the presence of indomethacin (Fig. 2). We also examined Fos expression in the presence of tetrodotoxin and found it unaffected (data not shown).
hrIL-1 induces COX-2 in enteric neurons. To investigate this observation further, we used immunohistochemistry with antibodies specific for COX-2 to examine the distribution of the inducible enzyme responsible for prostaglandin production whose activity might be reduced by indomethacin. Control preparations or preparations incubated with indomethacin had no labeling for COX-2. Incubation of preparations in hrIL-1
(10 ng/ml) caused the appearance of COX-2 immunoreactive cells in the myenteric and submucosal plexuses and also outside of the plexus (Fig. 5). The cells in plexuses had the shape, position, and characteristics of enteric neurons. Some of the cells outside of the plexus were immunoreactive for vimentin and so were likely to be interstitial cells of Cajal (31), but others were not, and these were not characterized further.
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Fos is expressed in specific subsets of enteric neurons. Having identified that Fos expression occurs in some, but not all enteric neurons, we examined which subsets of neurons were activated based on double-labeling studies with Fos and a series of neuronal markers that could be used in combination with the Fos antibody. Two concentrations of hrIL-1 were tested in these studies. Because the results were very similar (in terms of the proportion of double-labeled cells), data for the 10 ng/ml hrIL-1
are presented in Table 2.
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In the guinea pig ileum, four nonoverlapping markers can be used to account for 100% of the submucosal neurons (3). With the use of three of these (VIP, NPY, and calretinin) we established that hrIL-1 activated the majority of the VIP population of noncholinergic secretomotor neurons and some calretinin neurons (Fig. 4). No NPY neurons were Fos immunoreactive. Together these data account for
85% of the Fos expression induced with 10 ng/ml hrIL-1
, suggesting that Fos is also present in submucosal substance P-immunoreactive neurons, that could not be directly labeled with our approaches, because colchicine or culturing is required to visualize substance P in submucosal neurons.
In the colonic submucosal plexus, 55% of the total Fos neurons were VIP immunoreactive and 44% were nitric oxide synthase (NOS) immunoreactive (Fig. 4). Because VIP and NOS completely overlap (22), it appears that these are the same neurons. Why there was a slight difference in labeling is not clear. A small proportion of Fos-labeled cells were NPY neurons, and a few calbindin and calretinin neurons were also double-labeled, but 40% of the Fos immunoreactive neuronal nuclei did not label with the markers used in this study. Given the size of the calbindin, calretinin, and NPY populations it is surprising that this is so high, and it is not clear why some Fos immunoreactive neurons were not double labeled. One possibility is that the calbindin antibody used here was not as effective at labeling submucosal neurons.
In the myenteric plexus of the ileum and colon, 50% of the Fos immunoreactive neurons were also immunoreactive for NOS (Fig. 3). In the ileum, enkephalin immunoreactive neurons represented
30% of the Fos cells and smaller proportions were calbindin, calretinin, and somatostatin immunoreactive neurons. Given that some of these populations overlap to some extent, there may still be some cells not accounted for, but the majority of the Fos cells activated by hrIL-1
in the ileum can be accounted for by a limited number of functional subtypes of enteric neuron. In the colonic myenteric plexus, this is not the case, because only
50% of the activated neurons could be accounted for with these markers, suggesting that many activated neurons were likely to be in different functional classes than in the ileum.
Fos expression is increased in enteric glia after incubation with hrIL-1. In addition to neuronal Fos expression, enteric glia also expressed Fos when stimulated with hrIL-1
. In the myenteric plexus, there was no evidence of concentration dependency of this effect in which
10-20 glial cells were activated per ganglion (Table 3). In the submucosal plexus, it appeared that at higher concentrations of hrIL-1
, there was a greater response.
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The effect of indomethacin on enteric glial Fos expression in the myenteric plexus was less consistent than that for neuronal Fos expression, but significant reductions were nevertheless observed (Table 3). In the submucosal plexus, indomethacin appeared to largely block glial Fos expression, as was observed for neuronal Fos expression (Table 3).
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DISCUSSION |
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Previous studies (12, 20, 24, 26, 27, 32, 35, 37, 45) have used Fos expression as a marker of enteric neuronal activation in response to a variety of natural, innocuous, and noxious stimuli, including electrical nerve stimulation, mucosal stimulation, and inflammation and after the induction of intestinal anaphylaxis. Double-labeling studies similar to the present study have revealed that Fos can be used to map specific neuronal pathways that are activated in response to a given stimulus. We have previously used this technique to examine activation of submucosal pathways of the guinea pig ileum in response to PGE2 and found that VIP neurons were activated (10). Schicho et al. (37) found that myenteric NOS neurons were activated by a luminal acid challenge, as did Yuan and Yang (45) after insulin hyperglycemia was used to activate the myenteric plexus. Fos has also been used as an indicator of enteric glial activation. The range of stimuli reported to activate Fos in enteric glia include mucosal acid challenge of the stomach, stimulation of the central nervous system with a stable thyrotropin-releasing hormone analog RX-77368 (27), protease-activated receptor 2 agonists (16), as well as inflammatory mediators, as we have found in this study and others have shown before (34). It is not clear if the response of the enteric glia in the whole animal or isolated tissue studies is direct or indirect; however, studies from isolated/cultured enteric glia have demonstrated that IL-1 and protease-activated receptor 2 agonists cause upregulated c-fos mRNA and enhanced Fos expression, respectively (16).
As stated above, we previously showed that PGE2 activated a population of VIP immunoreactive secretomotor/vasomotor neurons in the guinea pig submucosal plexus. In that report, we found little evidence for activation of myenteric neurons, despite evidence that PGE2 can depolarize ileal myenteric neurons. We have extended those observations to show that IL-1 activates essentially the same population of submucosal neurons in the ileum and also in the colon. In addition, it appears that some additional neurons in the submucosal plexus of the ileum and colon are activated that are not immunoreactive for the neuronal markers used in this study. Given the size of the population in both cases, it seems most likely to be the proposed intrinsic primary afferent populations that express calbindin, tachykinins, and acetylcholine as described by Bornstein and Furness (3) and Kirchgessner et al. (20) for the ileum and Lomax and Furness for the colon (22). These neurons comprise
15 and 22% of the total cell population of the ileum and colon, respectively, and that is similar to what is not accounted for in this study. Although we used a calbindin antibody, it appears that either it failed to reveal this population, or that in combination with the Fos antibody, immunoreactivity was suppressed.
In the myenteric plexus of the ileum, we have identified the majority of neurons activated as belonging to classes of descending interneurons/motorneurons that express NOS or to interneurons/motorneurons that express enkephalin. In contrast, in the colon, many activated neurons express NOS, but few express enkephalin, and 50% of the activated neurons cannot be accounted for and so must be in other classes. It has been reported that calbindin neurons do not express Fos in response to distension, peristalsis, or forskolin and only weakly in response to electrical stimulation (32). However, Fos has been observed in AH-type calbindin neurons in the inflamed jejunum of animals infected with Trichinella spiralis (29). At this point, it is not clear if some of the uncharacterized neurons are calbindin neurons, and whether myenteric calbindin neurons express or do not express Fos when activated by inflammatory cytokines remains to be fully clarified.
The action of IL-1 has been investigated electrophysiologically by Kelles et al. (18, 19) and Xia et al. (44). In the guinea pig ileal submucosal plexus, Xia et al. (44) found that the majority of neurons classified as AH-type or S-type responded with a slow depolarization in a concentration-dependent manner to application of IL-1
. Kelles et al. (18) extended these observations to include the myenteric plexus and showed that only
20% of either AH-type or S-type neurons responded to application of IL-1
and that these responses were due to direct action on the enteric neurons. There is a good correlation between the electrophysiological data and that of the present study with regard to the myenteric plexus, although why Fos was observed in so few calbindin neurons (that would be expected to be of the AH-type electrophysiologically) is not clear. It is evident that there are major differences between submucosal and myenteric neurons in their ability to directly respond to IL-1
in the ileum. Kelles et al. (19) also examined the colonic myenteric plexus and, in contrast to the ileum and in agreement with the present study, found a far greater proportion of neurons that are directly activated. In that study, they also proposed that the responses to IL-1
were not mediated by PGE2, because the responses of single neurons were different when the two mediators were tested. This is not necessarily inconsistent with the current observations whose time course is quite different from that of an acute electrophysiological study, but further work is required to fully understand these observations.
The induction of COX-2 by IL-1 is suggested by the presence of COX-2 immunoreactivity in treated but not untreated tissues and by the sensitivity of the Fos expression to indomethacin treatment. COX-2 immunoreactivity has previously been observed in neurons of the myenteric plexus of the rat intestine after surgical manipulation (38) in the mouse stomach under basal conditions and in the inflamed human colon (31, 33). In the mouse stomach, COX-2 immunoreactivity has also been observed in vimentin immunoreactive cells that were shown to be interstitial cells of Cajal by Porcher et al. (31). In the present study, we did not find COX-2 immunoreactivity under basal conditions, suggesting in the guinea pig that this enzyme is not constitutively expressed as it is in the mouse. Consistent with our observations using Fos as a marker is that PGE2, one of the major products of COX-2, has an excitatory action on enteric neurons assessed by electrophysiological approaches (9, 13, 19). Interestingly, after chronic treatment with a stable analog of PGE2 the properties of these cells are altered in ways consistent with chronic states of activation seen in recordings from inflamed preparations (23).
In conclusion, we have found that the proinflammatory cytokine IL-1 specifically activates certain neurochemically defined neuronal populations in the guinea pig small and large intestine. Full functional significance of these data will require additional studies, by using chambered preparations that identify the sites of action of mediators at specific sites in the reflex pathways of the intestine. Nevertheless our data support a specific action of IL-1
on descending inhibitory motor neurons or interneurons expressing NOS, and so we might conclude that propagating motor activity would be altered in ileitis and colitis. This is in accord with previous functional studies (17, 41) that have examined propagating motility in vivo and the role of nitric oxide-mediated transmission in vitro. Our data also further highlight that activation of enteric glia is a feature of cytokine stimulation and support a role for glial cells in modulation of synaptic transmission or other processes in the enteric nervous system.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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