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Leukemia inhibitory factor mediates cytokine-induced suppression of myenteric neurotransmitter release from rat intestine
GASTROENTEROLOGY 1996;111:674–681
Leukemia Inhibitory Factor Mediates Cytokine-Induced Suppression of Myenteric Neurotransmitter Release From Rat Intestine GERT VAN ASSCHE and STEPHEN M. COLLINS Intestinal Diseases Research Programme, McMaster University, Hamilton, Ontario, Canada
Background & Aims: Exposure of rat longitudinal muscle myenteric plexus to the proinflammatory cytokine interleukin (IL) 1b mimics the effects of nematode infection on enteric nerve function through a hitherto unidentified protein intermediate. It is postulated that leukemia inhibitory factor (LIF), the downstream intermediate of several IL-1–induced neuroimmune interactions, mediates IL-1b –induced suppression of acetylcholine release from rat jejunum. Methods: Preparations were preloaded with [3H]choline, and [3H]acetylcholine release was induced by either electrical field stimulation or by 50 mmol/L KCl. Cytokines and anti-LIF antibodies were added to the preincubation media or to the superfusate before stimulation. Results: Human recombinant LIF had no immediate effects, but preincubation with the cytokine induced a concentration-dependent (2–100 ng/mL) and reversible suppression of acetylcholine release from rat longitudinal muscle myenteric plexus. The effects of human recombinant LIF on acetylcholine release were reversed by anti–human recombinant LIF–neutralizing antibody. Human recombinant IL-1b (10 ng/mL) induced a similar suppression of acetylcholine release, and the addition of anti-rat LIF antibody abolished the effects of exogenous IL-1 on acetylcholine release. Conclusions: IL-1b suppresses neurotransmitter release from rat myenteric plexus via the induction of LIF as a downstream intermediate.
I
nflammation of the gastrointestinal tract results in disturbance of motility in humans and in animal models. In human inflammatory bowel disease, the disruption of normal motor patterns contributes to commonly observed clinical features such as diarrhea and abdominal cramping. Enteric nerves innervating the muscle layers from the myenteric and submucosal plexus play an important role in the generation of normal motility patterns and secretion, as well as in mediating sensation from the gut. Therefore, disruption of their structural and functional integrity will inevitably lead to intestinal motor and secretory dysfunction and to altered sensory perception. Structural alterations of enteric nerves have been / 5e11$$0013
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documented widely in human inflammatory bowel disease and consist of both degenerative and hypertrophic changes of the myenteric plexus.1,2 Functional enteric nerve changes in the inflamed intestine have been studied more extensively in experimental enteritis. Inflammation induced in the small intestine by nematode infection of the rat results in myenteric nerve dysfunction,3,4 and similar results have been obtained recently in rats with colitis induced by trinitrobenzene sulfonic acid, indicating that this is a response to inflammation that is not specific for the manner in which the inflammation is induced.5 Inflammatory mediators produced in situ or systemically generate structural and functional changes in the central and peripheral nervous system.6 Previous studies in our laboratory have identified the proinflammatory cytokine interleukin (IL) 1b as a putative mediator of myenteric nerve dysfunction in intestinal inflammation. The expression of IL-1b messenger RNA and protein are both increased in the neuromuscular layers of the inflamed small intestine at an early stage after nematode infection in the rat.7 In addition, exogenous IL-1b suppresses acetylcholine and norepinephrine release from rat jejunal myenteric nerves in vitro.8 – 10 Treatment of infected rats with an antagonist to IL-1 abrogated the change in neurotransmitter release, indicating a causal role for this cytokine in mediating altered neuronal function in this model. The suppression of myenteric neurotransmitter release by IL-1b requires the induction of a protein intermediate,8,9 which has not been further characterized. Prime candidates for mediating the action of IL-1b in the context are members of the recently described neuropoietic cytokine family, including IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary nerve trophic factor, and oncostatin M.11 In autonomic ganglia, LIF has been identified Abbreviations used in this paper: EFS, electrical field stimulation; hr, human recombinant; IL, interleukin; LIF, leukemia inhibitory factor; LMMP, longitudinal muscle myenteric plexus. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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as the downstream mediator for the action of IL-1b on neuronal development and injury-induced alterations.12,13 The source of LIF in cervical ganglia is considered to be the neuroglia and other support cells rather than the nerves. In the gastrointestinal tract, enteric glial cells have been shown to produce IL-6, which shares several biological effects with LIF.14 The purpose of the present study was to investigate the role of LIF as a downstream mediator of the IL-1b– induced cholinergic nerve dysfunction in a previously characterized longitudinal muscle myenteric nerve preparation from the rat jejunum.3 Human recombinant (hr) LIF and hrIL-1b were used in all experiments. The amino acid sequence of rat, murine, and human LIF shows an 80% homology, and the bioactive NH2 terminal is identical in the three species.15 Both hrLIF and hrIL-1b are highly effective in rat nervous tissue.12 Acetylcholine release was studied as a parameter of myenteric nerve function because previous work has shown that the release of this intrinsic neurotransmitter is impaired dramatically by exogenous IL-1b.8
Materials and Methods Animals Male Sprague–Dawley rats (weight range, 180–200 g) were obtained from the Charles River Breeding Laboratories (St. Constant, Quebec, Canada). Specific pathogen-free animals were housed in filtered cages on a 12-hour light-dark cycle. Sterilized food and water were supplied ad libitum. Rats (weight range, 250–350 g) were killed by rapid deceleration and cervical dislocation. All protocols were approved by the McMaster Animal Ethics Committee.
Materials Hemicholinium-3 was obtained from Sigma Chemical Co. (St. Louis, MO); [methyl-3H]choline (sp act, 80 Ci/mmol) was obtained from New England Nuclear Corp. (Boston, MA); NCS-II tissue solubilizer was obtained from Amersham Corp. (Arlington Heights, IL); and hrLIF, hrIL-1b, monoclonal anti– hrIL-1b, and goat polyclonal anti-hrLIF antibody were purchased from R&D Systems (Minneapolis, MN). Purified rabbit anti-rat LIF antibody was a kind gift from Dr. P. H. Patterson (California Institute of Technology, Pasadena, CA). All other chemicals were of analytical grade.
Tissue Preparation The longitudinal muscle myenteric plexus (LMMP) preparation was obtained from normal, specific pathogen–free rats as described previously.3 Briefly, after midline laparotomy, the proximal jejunum was removed quickly, and segments were placed carefully on a 4-mm glass pipette. The serosal surface was scored with a dulled scalpel blade and the LMMP layer was gently peeled off with a wet cotton swab. The integrity of the LMMP was checked using light microscopy.
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Measurement of Acetylcholine Release Acetylcholine release was measured as described previously.3 Briefly, to obtain LMMP loaded with labeled acetylcholine, tissues were incubated with 0.2 mmol/L [methyl3 H]choline in Krebs’–Ringer’s solution containing the following (in mmol/L): NaCl, 120.9; KCl, 5.9; CaCl2 , 2.5; MgCl2 , 1.2; NaHCO3 , 15.5; NaH2PO4 , 1.2; and glucose, 11.1 for 1 hour at 377C and were oxygenated with 95% O2 and 5% CO2 . Tissues were washed subsequently twice in 5 mL of normal Krebs’ buffer and suspended in a water-jacketed superfusion chamber maintained at 377C using a thermostatically controlled water pump (Haake, Karlsruhe, Germany). The LMMP were superfused with oxygenated Krebs’ solution containing 10 mmol/L hemicholinium-3 at a flow rate of 1 mL/min using a peristaltic pump (Buchler, Fort Lee, NJ). Superfusate was collected at 2-minute intervals using an Altrorac 7000 fraction collector (LKB, Bromma, Sweden). After a 40-minute equilibration period, tritium release was stimulated by electrical field stimulation (EFS) and by a 50 mmol/L KCl-Krebs’ solution at 58 minutes. EFS was applied via two parallel silver electrodes at 10 Hz, 0.5 milliseconds, and 30 V for 1 minute. These parameters have been shown previously to optimally release [3H]acetylcholine from rat jejunal LMMP and to reflect stimulation of enteric neurons.3 After completing the protocol, 4 mL of aqueous scintillation fluid ACS (Amersham Corp., Oakville, Ontario, Canada) was added to each vial containing 2 mL of superfusate, and the tritium content was determined by a Beckman liquid scintillation counter (model LS 5801; Beckman Instruments, Inc., Fullerton, CA) with a counting efficiency of 35%. The tissues were blotted dry, weighed, and dissolved overnight in 1 mL of NCS-II tissue solubilizer. The solution was neutralized with 100 mL of glacial acetic acid. Four milliliters of scintillation fluid was added, and the tritium content was counted as described above to determine the remaining amount of radioactivity in the tissue. The evoked release of 3H on stimulation was expressed as the fractional release of the tritium present in the tissue at the time of stimulation as described previously.3 In an earlier study using high-performance liquid chromatography, we have shown that the evoked tritium release consists entirely of [3H]acetylcholine and therefore is an accurate reflection of functional neurotransmitter released during stimulation.3
Protocols Involving hr Cytokines To study immediate and delayed effects of hrLIF on H release, hrLIF at different concentrations (0.02–100 ng/ mL) was added directly to the superfusion buffer or to the preincubation media at various time points before onset of superfusion. Control tissues received the same volume of saline at equal time points. hrIL-1b was added to the incubation buffer 90 minutes before starting the superfusion at a concentration of 10 ng/mL. These parameters have been proven previously to result in a maximal suppression of acetylcholine release from rat jejunal LMMP.8 To study the specificity of hrLIF–mediated effects, the cytokine was incubated with an equal volume of 1 mg/mL goat polyclonal hrLIF-neutralizing 3
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antibody for 20 minutes at 377C. Twenty-milliliter aliquots of this mixture were added to the preincubation media for 90 minutes, resulting in a final hrLIF concentration of 20 ng/mL. Controls were treated with a mixture of sterile phosphatebuffered saline and hrLIF at an equal concentration.
Data Analysis All studies consisted of at least four separate experiments involving different animals. The data are expressed as mean { SE. For normally distributed data, Student’s t test was used to compare two groups, and one-way analysis of variance was used when comparing three or more groups. For nonparametric data, Mann–Whitney rank sum test and Kruskal–Wallis analysis of variance were applied, respectively. Statistical significance was inferred at P values of õ0.05.
Results hrLIF Effects on Acetylcholine Release To examine immediate effects of LIF on basal or stimulated acetylcholine release, hrLIF (final concentrations, 2 ng/mL and 20 ng/mL) was added directly to the superfusion solution during the equilibration period or 8 minutes before EFS or KCl stimulation. Addition of hrLIF directly to the superfusate solution did not cause any changes in basal or stimulated outflow of tritium from the LMMP (data not shown). Delayed effects of hrLIF on acetylcholine release were determined by incubating the tissues in oxygenated Krebs’ buffer at 377C 60 minutes before [methyl-3H]choline administration, resulting in a total incubation time of 120 minutes. hrLIF was added to the incubating LMMP at various time points. When added to the preincubation media 90 minutes before the onset of superfusion, hrLIF caused
Figure 2. Time course of hrLIF-induced suppression of 3H release. Tissues were preincubated for 120 minutes before superfusion. hrLIF (20 ng/mL) was added at indicated time points during the preincubation. 3H release was stimulated by EFS (h) or KCl (/). Data are expressed as the percentage of the stimulation observed after incubation with saline (control release). Values are expressed as mean { SE of four independent experiments. *Value significantly different from control (P õ 0.05).
a marked, dose-dependent suppression of acetylcholine release (Figure 1). A significant suppression of EFS-stimulated release occurred at hrLIF concentrations from 2 to 100 ng/mL (2 ng/mL: 52.0% { 16.8% of control fractional 3H release, P õ 0.05; 20 ng/mL: 23.4% { 9.7%, P õ 0.01; and 100 ng/mL, 39.4% { 13.3%, P õ 0.01). Similar results were obtained for KCl-evoked acetylcholine release (20 ng/mL: 32.3% { 6.0%, P õ 0.01; 100 ng/mL: 55.4% { 21.9% of control fractional 3 H release, P õ 0.05). The maximally effective concentration for LIF-induced suppression of acetylcholine release induced by either EFS or KCl was 20 ng/mL. As shown in Figure 2, a minimal incubation time of 60 minutes was required for a significant suppression of EFS-evoked acetylcholine release (36.2% { 0.15% of control fractional 3H release; P õ 0.01). Maximal suppression occurred after 90 minutes of preincubation (23.4% { 10.6% of control fractional 3H release; P õ 0.01). Impairment of KCl-evoked release only reached significance after 90 minutes of preincubation. Specificity of hrLIF Effects
Figure 1. Concentration dependence of hrLIF-induced suppression of 3 H release from rat LMMP preparations. Tissues were incubated for 90 minutes with saline or hrLIF in the concentrations indicated for 90 minutes before subsequent stimulation by EFS (h) or KCl (/). The release of acetylcholine was measured as 3H release and expressed as a percentage of the release obtained in the absence of LIF. Shown are mean { SE from four experiments. *Value significantly different from control (P õ 0.05).
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Using the optimal conditions for hrLIF suppression of 3H release (20 ng/mL and 90 minutes) as determined earlier, we repeated the cytokine incubation in a separate set of tissues. Specific hrLIF-neutralizing antibody (10 mg/mL) added to hrLIF before and during preincubation abolished the effects of hrLIF on stimulated 3 H release (Figure 3). Values for EFS- (1.53% { 0.45%) and KCl-evoked (1.01% { 0.17%) releases when incubated with hrLIF and antibody combined were not significantly different from controls (control EFS, 1.22% { 0.13%; KCl, 0.88% { 0.11% fractional 3H release), WBS-Gastro
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Figure 3. Effect of hrLIF-neutralizing antibody on LIF-induced suppression of EFS-evoked 3H release. LMMP preparations were incubated for 90 minutes with saline (h), hrLIF (20 ng/mL) ( ), hrLIF plus antibody (AB) (10 mg/mL) (j), or antibody alone ( ). Values are expressed as mean { SE from four experiments. *Value significantly different from control (P õ 0.001).
whereas hrLIF alone caused a significant suppression of the evoked 3H release (EFS: 0.22% { 0.07% fractional 3 H release, P õ 0.001; KCl: 0.30% { 0.06%, P õ 0.05). hrLIF-neutralizing antibody alone did not cause any significant changes in 3H release (Figure 3). Reversibility of hrLIF Effects To investigate the reversibility of the impaired H release by hrLIF, tissues were incubated with the cytokine (20 ng/mL) for 90 minutes, washed twice in normal Krebs’ buffer, and allowed to recover for 120 minutes in normal Krebs’ buffer before the onset of superfusion. As shown in Figure 4, the suppression of 3H release by hrLIF (20 ng/mL) was reversed by this washout procedure. EFS-stimulated release (hrLIF, 0.90% { 0.21%; vs. control, 0.89% { 0.19% fractional 3H release) and KCl-evoked release (hrLIF, 0.65% { 0.14%; vs. controls, 3
Figure 4. Reversibility of hrLIF effects on 3H release when stimulated by EFS (j) or KCl ( ). Tissues were incubated with saline or LIF (20 ng/mL), followed by a 60-minute incubation in normal Krebs’ buffer and 60-minute incubation with [3H]choline. Values are expressed as mean { SE of four independent experiments. cont, control.
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Figure 5. Effect of rat LIF-neutralizing antibody on IL-1b–induced suppression of (A ) EFS- and (B ) KCl-stimulated 3H release. Tissues were incubated with saline (h), IL-1b ( ), IL-1b plus antibody (AB) (j), or antibody alone ( ). Data are expressed as mean { SE from four experiments. *Significantly different from control (P õ 0.05).
0.49% { 0.09% fractional 3H release) were not significantly different in controls and hrLIF-incubated tissues. Interactions Between IL-1 and Endogenous LIF Incubation of LMMP with 10 ng/mL of hrIL-1 for 90 minutes resulted in a significant suppression of stimulated 3H release (EFS: IL-1b, 0.28% { 0.12%; vs. controls, 1.24% { 0.17%; P õ 0.01; KCl: IL-1b, 0.42% { 0.12%; vs. controls, 0.88% { 0.12% fractional 3H release; P õ 0.05) (Figure 5). Previous studies have shown that these conditions are optimal for IL-1–induced suppression of 3H release from myenteric plexus in rat jejunum.8 Addition of polyclonal anti-rat LIF antibody (20 mg/mL) to the incubation media abolished the effects of hrIL-1b on EFS- and KCl-evoked 3H release from the LMMP (Figure 5). EFS-evoked 3H release after incubation with IL-1b (10 ng/mL) and anti-rat LIF antibody was 1.02% { 0.09% (% fractional 3H release) vs. 1.24% { 0.17% in controls. Similarly the KCl-evoked release after exposure to IL-1 and anti-rat LIF antibody totaled 0.82% { 0.16% (fractional 3H release) vs. 0.87% { 0.11% in controls. Anti-rat LIF antibody alone did not cause any significant changes in 3H release. Addition WBS-Gastro
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Table 1. [3H]Choline Uptake of LMMP Preparations After Preincubation With hrLIF hrLIF (ng/mL)
0
0.2
2
20
100
[3H]choline uptake (1103 dpm/mg )
9.85 { 0.85
5.94 { 1.02
7.45 { 0.43
6.95 { 0.34
7.63 { 1.85
NOTE. hrLIF at different concentrations was added 90 minutes before onset of superfusion. [3H]choline uptake was calculated as the sum of the total 3H released during the experiment and the residual 3H in the tissue. Results were normalized for the weight of each LMMP. Values are expressed as mean { SE from four independent experiments. No significant differences (P ú 0.05) were observed between treatment groups using analysis of variance.
of anti-hrLIF antibody to the hrIL-1b preincubation did not interfere with IL-1b–induced suppression of 3H release (EFS, 0.31% { 0.07% vs. 1.24% { 0.17% control fractional 3H release; P õ 0.05; not significantly different from IL-1b alone). As an additional external control, IL1 incubation was repeated in the presence of normal rabbit serum (1:200 dilution). Addition of normal rabbit serum did not influence the effects of hrIL-1 on 3H release from LMMP (data not shown). Effects of hrLIF on 3H Content of the LMMP The total uptake of [methyl-3H]choline, as determined by the sum of 3H released from the LMMP and the residual 3H remaining in the tissue at the end of the experiment, per milligram of tissue weight was not significantly altered by hrLIF at different doses (Table 1). However, incubation of the tissue with IL-1b (10 ng/ mL) resulted in a significant suppression of [methyl3 H]choline uptake (IL-1b, 6.16 { 0.53 dpm/mg; control, 10.03 { 0.87 1 103 dpm/mg; P õ 0.01; n Å 5).
Discussion The results of the present study provide circumstantial evidence to support a role for the neuropoietic cytokine LIF as a downstream mediator of the suppressive effects of IL-1 on the myenteric plexus of the rat intestine. Previous studies in our laboratory have shown that the suppression of enteric neurotransmitter release by proinflammatory cytokines requires the induction of protein intermediates.8,9 However, no further data were obtained about the nature of these downstream mediators, and to the best of our knowledge, there are no reports available in the literature on secondary mediators of the actions of IL-1 on enteric cholinergic nerve function. The role of immune-regulatory cytokines in neuronal development and their response to injury has been characterized most extensively in autonomic ganglia of the rat.6,16 Normal neuronal maturation and injurious alterations are closely regulated by a variety of growth factors and cytokines. A key position in these neuroimmune interactions is occupied by an emerging family of cytokines that share several biological actions on neuronal and / 5e11$$0013
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nonneuronal cells through a common signal transduction pathway, the transmembrane glycoprotein gp130.17,18 From this group of cytokines, ciliary neurotrophic factor, cholinergic differentiation factor or LIF, IL-6, and IL-11, are thought to play an important role in neuroimmune interactions. LIF, originally discovered as a differentiation and inhibition factor of murine myeloma cell lines,19 has unique properties in various nerve types. It improves the maturation and survival of spinal motor neurons and sensory neurons from the dorsal root ganglia,20,21 it induces cholinergic phenotypic switch and neuropeptide expression in sympathetic nerve cultures,12 and it is involved in neuronal response to injury in vivo.22,23 In sympathetic ganglia, LIF has been shown to act as a soluble intermediate for the IL-1– and tumor necrosis factor a–induced expression of preprotachykinin messenger RNA and substance P.12,24 In the present study, we provide evidence that the cytokine cascade of LIF induction by IL-1 is probably not restricted to the sympathetic cervical ganglia but may be an important sequence in the observed injury-related alterations of intestinal nerve function. In our in vitro system, the action of LIF on cholinergic nerve function occurs at concentrations between 2 and 100 ng/mL (88 pmol/L to 4.4 nmol/L). Similar concentrations are required for the induction of substance P and choline-acetyl transferase, as well as for effects on cell survival in cultured sympathetic ganglia and somatic motor neurons.15,21,25,26 The concentrations of hrLIF required to suppress rat myenteric neurotransmitter release in the present study are also consistent with the reported affinity constant of the LIF receptor in murine cell lines (dissociation constant of 30–100 pmol/L).27 A different dose-response relation was observed for the impairment of KCl-induced 3H release by hrLIF compared with the EFS-evoked release from rat myenteric nerves. Earlier work in our laboratory has shown that different membrane-localized mechanisms are involved in KCl- and EFS-evoked 3H release in the rat LMMP superfusion system because the EFS-induced but not the KCl-induced release was totally inhibited by the axonal sodium channel inhibitor tetrodotoxin.3 Thus, differences in neurotransmitter release mechanisms triggered by EFS and WBS-Gastro
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KCL probably account for a different hrLIF sensitivity. The reversal of IL-1b suppression of acetylcholine release by specific anti-rat LIF antibody shows an interaction between these two cytokines in the myenteric plexus. The anti-rat LIF antibody used in this study was raised by immunizing rabbits with a synthetic peptide corresponding to the NH2 -terminal 11 amino acids of rat LIF; this was followed by purification of the antiserum based on its affinity for the synthetic peptide.15,28 This part of the native molecule is responsible for the bioactivity of LIF on sympathetic neurons.15 Previous studies have shown that this antibody lacks cross-reactivity with other neuropoietic cytokines, such as ciliary neurotrophic factor and IL-6.29 Furthermore, normal rabbit serum did not interfere with the IL-1–induced effects on neurotransmitter release in rat LMMP. Therefore, it is highly unlikely that the antibody neutralized the activity of a molecule other than LIF. In contrast, the lack of inhibition of hrIL-1b–mediated effects on rat intestinal LMMP by polyclonal anti–hrLIF most likely reflects differences between the amino acid sequences of human and rat LIF. The cascade of IL-1b induction of LIF as the downstream mediator for its effects on neurons was originally described in sympathetic ganglia and in bone marrow.13,30 We chose to investigate the effects of LIF on cholinergic myenteric nerve function because these neurons constitute a major intrinsic population in the myenteric plexus, and we have shown previously that IL-1– induced suppression of acetylcholine release from LMMP requires de novo protein synthesis. In contrast, previous studies from our group have shown that exogenous IL1b induces decreased norepinephrine release from rat myenteric plexus at least partially through the autoinduction of IL-1.9,31 The ability of LIF to suppress acetylcholine release in the present study does not reflect a reduction in the uptake of [3H-methyl]choline by the myenteric nerves in the LMMP. However, in myenteric plexus obtained from Trichinella spiralis–infected rat jejunum, suppression of acetylcholine release is accompanied by a reduced uptake of [3H-methyl]choline.3 Both alterations in myenteric nerve function were reproduced by exogenous hrIL-1b in normal rat LMMP in a previous study from our group,8 and this finding was confirmed in the present study. Therefore, it is unlikely that LIF accounts for all the changes in cholinergic nerve function attributed to IL-1 in the parasitized rat jejunum. In contrast to our results, comparable concentrations of LIF have been shown to increase acetylcholine content by up-regulating choline acetyltransferase in cultured sympathetic ganglia and motor neurons.15,21,32,33 However, these nerve cul/ 5e11$$0013
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tures were obtained from embryonic animals and did not show a cholinergic phenotype before exposure to LIF as opposed to the mature cholinergic enteric nerves present in the adult rat LMMP. Moreover, the induction of choline acetyltransferase in cultured embryonic nerves required several days of incubation with LIF compared with the 60 minutes in our system. The cellular source of LIF in the myenteric plexus is not clear. Various cell types are known to produce LIF in vitro when the appropriate stimulus is provided. Cultured fibroblasts and brain astroglia can be induced to secrete LIF,19,34 and rat peritoneal mast cells produce LIF in vitro when stimulated with a calcium ionophore.35 However, in normal rat LMMP, glial cells, macrophagelike cells, and fibroblasts are the most likely source of LIF because peritoneal mast cells are sparse in these preparations. Evidence implicating neuroglia as the source of LIF has been found in cultured sympathetic ganglia. In these mixed nerve cultures, IL-1–induced LIF secretion was eliminated when cultures were purified by antimitotic agents to eliminate nonneuronal foster cells.13,24 Furthermore, glial cells in culture are capable of producing LIF,13,34,36 and in situ studies have shown that peripheral nerve section induces increased LIF expression by Schwann cells.37 In the intestine, previous work in our laboratory has shown that hrIL-1b induces ultrastructural changes in macrophage-like cells of the myenteric plexus suggestive of phagocytosis and secretion.31 Nevertheless, we cannot exclude entirely a direct action of exogenous IL-1b on neuronal IL-1 receptors to induce LIF exerting an autocrine effect because neuronal IL-1 receptors have been described in the central nervous system.38 – 41 The functional relevance of cytokine-mediated effects on myenteric nerve function is supported by the recent report from our laboratory of copious in situ expression of IL-1b messenger RNA and increases in IL-1b protein in the jejunal LMMP region of nematode-infected rats.7 Contrary to IL-1b, constitutive LIF expression is not readily detectable in most tissues except for the pregnant uterus,42,43 but LIF appears to be up-regulated in inflammatory states. Elevated plasma levels of bioactive LIF have been reported in septic shock and synovial fluid of patients with arthritis has been found to contain significant amounts of LIF, similar to the concentrations that were required for the effects on enteric nerves in our study.44,45 In summary, we have shown that LIF impairs cholinergic nerve function in isolated rat myenteric plexus. Furthermore, the results from the present study provide evidence for the induction of LIF as a downstream mediator for the action of IL-1b on myenteric nerve function. Taken together, these findings extend the role of neuroWBS-Gastro
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poietic cytokines, such as LIF, as important modulators of the enteric nervous system. Further research will be required to establish the role of LIF in injury-related nerve dysfunction associated with experimental and clinical intestinal inflammation.
20.
21.
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factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. J Biol Chem 1984;259:10978–10982. Murphy M, Reid K, Hilton DJ, Barlett PF. Generation of sensory neurons is stimulated by leukemia inhibitory factor. Proc Natl Acad Sci USA 1991;88:3498–3501. Martinou JC, Martinou I, Kato AC. Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motor neurons in vitro. Neuron 1992;8:737–744. Rao MS, Sun Y, Escary JL, Perreau J, Tresser S, Patterson PH, Zigmond RE, Brulet P, Landis SC. Leukemia inhibitory factor mediates an injury response but not a target directed developmental transmitter switch in sympathetic neurons. Neuron 1993;11: 1175–1185. Li M, Sendtner M, Smith A. Essential function of LIF receptor in motor neurons. Nature 1995;378:724–727. Ding M, Hart RP, Jonakait GM. Tumor necrosis factor–a induces substance P in sympathetic ganglia through the sequential induction of interleukin-1 and leukemia inhibitory factor. J Neurobiol 1995;28:445–454. Lewis SE, Rao MS, Symes AJ, Dauer WT, Fink JS, Landis SC, Hyman SE. Coordinate regulation of choline acetyltransferase, tyrosine hydroxilase and neuropeptide mRNAs by ciliary neurotrophic factor and leukemia inhibitory factor in cultured sympathetic neurons. J Neurochem 1994;63:429–438. Kessler JA, Ludlam WH, Freidin MM, Hall DH, Michaelson MD, Spray DC, Dougherty M, Batter DK. Cytokine-induced programmed death of cultured sympathetic neurons. Neuron 1993; 11:1123–1132. Hilton DJ, Nicola NA, Metcalf D. Distribution and comparison of receptors for leukemia inhibitory factor on murine hematopoietic and hepatic cells. J Cell Physiol 1991;146:207–215. Fukada K, Rushbrook JI, Towle MF. Immunoaffinity purification and dose-response of cholinergic neuronal differentiation factor. Dev Brain Res 1991;62:203–214. Ulich TR, Fann MJ, Patterson PH, Williams JH, Samal B, Del Castillo J, Yin S, Guo K, Remick DG. Intratracheal injection of LPS and cytokines. V. LPS induces expression of LIF and LIF inhibits acute inflammation. Am J Physiol 1994;267:L442– L446. Wetzler M, Talpaz M, Lowe DG, Baiocchi G, Gutterman JU, Kurzrock R. Constitutive expression of leukemia inhibitory factor RNA by human bone marrow stromal cells and modulation by IL-1, TNF-a and TGF-b. Exp Hematol 1991;19:347–351. Ru¨hl A, Berezin I, Collins SM. Involvement of eicosanoids and macrophage-like cells in cytokine-mediated changes in rat myenteric nerves. Gastroenterology 1995;109:1852–1862. Zurn AD, Werren F. Development of CNS cholinergic neurons in vitro: selective effects of CNTF and LIF on neurons from mesencephalic cranial motor nuclei. Dev Biol 1994;163:309–315. Michikawa M, Kikuchi S, Kim SU. Leukemia inhibitory factor (LIF) mediated increase of choline acetyltransferase activity in mouse spinal cord neurons in culture. Neurosci Lett 1992;140:75–77. Wesselingh SL, Cough NM, Finlay-Jones JJ, McDonald PJ. Detection of cytokine mRNA in astrocyte cultures using the polymerase chain reaction. Lymphokine Cytokine Res 1990;9:177–185. Marshall JS, Gauldie J, Nielsen L, Bienenstock J. Leukemia inhibitory factor production by rat mast cells. Eur J Immunol 1993;23: 2116–2120. Patterson PH, Chun LLY. The influence of non-neuronal cells on catecholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons. Proc Natl Acad Sci USA 1974;71:3607–3610. Banner LR, Patterson PH. Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia. Proc Natl Acad Sci USA 1994;91:7109–7113.
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38. Farrar WL, Kilian PL, Ruff MR, Hill JM, Pert CB. Visualization and characterization of interleukin 1 receptors in brain. J Immunol 1987;139:1902–1910. 39. Farrar WL, Kilian PL, Ruff MR, Hill JM, Pert CB. Characterization of interleukin 1 receptors in brain. Adv Biochem Psychopharmacol 1988;21:487–500. 40. Katsuura G, Gotschall PE, Arimura A. Identification of a high affinity receptor for interleukin-1 in rat brain. Biochem Biophys Res Commun 1988;156:61–67. 41. Takao T, Tracey DE, Mitchell WM, De Souza EB. Interleukin-1 receptors in mouse brain: characterization and neuronal localization. Endocrinology 1990;127:3070–3078. 42. Croy BA, Guilbert LJ, Browne MA, Cough NM, Stinchcomb DT, Reed N, Wegmann TG. Characterization of cytokine production by the metrial gland and granulated metrial gland cells. J Reprod Immunol 1991;19:149–166. 43. Metcalf D. The leukemia inhibitory factor (LIF). Int J Cell Cloning 1991;9:95–108. 44. Warring P, Wycherley K, Cary D, Nicola N, Metcalf D. Leukemia
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inhibitory factor levels are elevated in septic shock and various inflammatory body fluids. J Clin Invest 1992;90:2031–2037. 45. Warring PM, Carroll GJ, Kandiah DA, Buirski G, Metcalf D. Increased levels of leukemia inhibitory factor in synovial fluid from patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 1993;36:911–915.
Received February 6, 1996. Accepted May 24, 1996. Address requests for reprints to: Stephen M. Collins, M.D., Division of Gastroenterology, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. Fax: (905) 521-4958. Supported by a grant from the Medical Research Council of Canada (to S.M.C.) and by grants from the National Fonds voor Wetenschappelijk Onderzoek and D. Collen Research Foundation, Belgium (to G.V.A.). The authors thank Dr. P. H. Patterson (California Institute of Technology, Pasadena, CA) for his kind gift of purified rabbit anti-rat leukemia inhibitory factor antibody.
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Leukemia Inhibitory Factor Mediates Cytokine-Induced Suppression of Myenteric Neurotransmitter Release From Rat Intestine GERT VAN ASSCHE and STEPHEN M. COLLINS Intestinal Diseases Research Programme, McMaster University, Hamilton, Ontario, Canada
Background & Aims: Exposure of rat longitudinal muscle myenteric plexus to the proinflammatory cytokine interleukin (IL) 1b mimics the effects of nematode infection on enteric nerve function through a hitherto unidentified protein intermediate. It is postulated that leukemia inhibitory factor (LIF), the downstream intermediate of several IL-1–induced neuroimmune interactions, mediates IL-1b –induced suppression of acetylcholine release from rat jejunum. Methods: Preparations were preloaded with [3H]choline, and [3H]acetylcholine release was induced by either electrical field stimulation or by 50 mmol/L KCl. Cytokines and anti-LIF antibodies were added to the preincubation media or to the superfusate before stimulation. Results: Human recombinant LIF had no immediate effects, but preincubation with the cytokine induced a concentration-dependent (2–100 ng/mL) and reversible suppression of acetylcholine release from rat longitudinal muscle myenteric plexus. The effects of human recombinant LIF on acetylcholine release were reversed by anti–human recombinant LIF–neutralizing antibody. Human recombinant IL-1b (10 ng/mL) induced a similar suppression of acetylcholine release, and the addition of anti-rat LIF antibody abolished the effects of exogenous IL-1 on acetylcholine release. Conclusions: IL-1b suppresses neurotransmitter release from rat myenteric plexus via the induction of LIF as a downstream intermediate.
I
nflammation of the gastrointestinal tract results in disturbance of motility in humans and in animal models. In human inflammatory bowel disease, the disruption of normal motor patterns contributes to commonly observed clinical features such as diarrhea and abdominal cramping. Enteric nerves innervating the muscle layers from the myenteric and submucosal plexus play an important role in the generation of normal motility patterns and secretion, as well as in mediating sensation from the gut. Therefore, disruption of their structural and functional integrity will inevitably lead to intestinal motor and secretory dysfunction and to altered sensory perception. Structural alterations of enteric nerves have been / 5e11$$0013
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documented widely in human inflammatory bowel disease and consist of both degenerative and hypertrophic changes of the myenteric plexus.1,2 Functional enteric nerve changes in the inflamed intestine have been studied more extensively in experimental enteritis. Inflammation induced in the small intestine by nematode infection of the rat results in myenteric nerve dysfunction,3,4 and similar results have been obtained recently in rats with colitis induced by trinitrobenzene sulfonic acid, indicating that this is a response to inflammation that is not specific for the manner in which the inflammation is induced.5 Inflammatory mediators produced in situ or systemically generate structural and functional changes in the central and peripheral nervous system.6 Previous studies in our laboratory have identified the proinflammatory cytokine interleukin (IL) 1b as a putative mediator of myenteric nerve dysfunction in intestinal inflammation. The expression of IL-1b messenger RNA and protein are both increased in the neuromuscular layers of the inflamed small intestine at an early stage after nematode infection in the rat.7 In addition, exogenous IL-1b suppresses acetylcholine and norepinephrine release from rat jejunal myenteric nerves in vitro.8 – 10 Treatment of infected rats with an antagonist to IL-1 abrogated the change in neurotransmitter release, indicating a causal role for this cytokine in mediating altered neuronal function in this model. The suppression of myenteric neurotransmitter release by IL-1b requires the induction of a protein intermediate,8,9 which has not been further characterized. Prime candidates for mediating the action of IL-1b in the context are members of the recently described neuropoietic cytokine family, including IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary nerve trophic factor, and oncostatin M.11 In autonomic ganglia, LIF has been identified Abbreviations used in this paper: EFS, electrical field stimulation; hr, human recombinant; IL, interleukin; LIF, leukemia inhibitory factor; LMMP, longitudinal muscle myenteric plexus. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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as the downstream mediator for the action of IL-1b on neuronal development and injury-induced alterations.12,13 The source of LIF in cervical ganglia is considered to be the neuroglia and other support cells rather than the nerves. In the gastrointestinal tract, enteric glial cells have been shown to produce IL-6, which shares several biological effects with LIF.14 The purpose of the present study was to investigate the role of LIF as a downstream mediator of the IL-1b– induced cholinergic nerve dysfunction in a previously characterized longitudinal muscle myenteric nerve preparation from the rat jejunum.3 Human recombinant (hr) LIF and hrIL-1b were used in all experiments. The amino acid sequence of rat, murine, and human LIF shows an 80% homology, and the bioactive NH2 terminal is identical in the three species.15 Both hrLIF and hrIL-1b are highly effective in rat nervous tissue.12 Acetylcholine release was studied as a parameter of myenteric nerve function because previous work has shown that the release of this intrinsic neurotransmitter is impaired dramatically by exogenous IL-1b.8
Materials and Methods Animals Male Sprague–Dawley rats (weight range, 180–200 g) were obtained from the Charles River Breeding Laboratories (St. Constant, Quebec, Canada). Specific pathogen-free animals were housed in filtered cages on a 12-hour light-dark cycle. Sterilized food and water were supplied ad libitum. Rats (weight range, 250–350 g) were killed by rapid deceleration and cervical dislocation. All protocols were approved by the McMaster Animal Ethics Committee.
Materials Hemicholinium-3 was obtained from Sigma Chemical Co. (St. Louis, MO); [methyl-3H]choline (sp act, 80 Ci/mmol) was obtained from New England Nuclear Corp. (Boston, MA); NCS-II tissue solubilizer was obtained from Amersham Corp. (Arlington Heights, IL); and hrLIF, hrIL-1b, monoclonal anti– hrIL-1b, and goat polyclonal anti-hrLIF antibody were purchased from R&D Systems (Minneapolis, MN). Purified rabbit anti-rat LIF antibody was a kind gift from Dr. P. H. Patterson (California Institute of Technology, Pasadena, CA). All other chemicals were of analytical grade.
Tissue Preparation The longitudinal muscle myenteric plexus (LMMP) preparation was obtained from normal, specific pathogen–free rats as described previously.3 Briefly, after midline laparotomy, the proximal jejunum was removed quickly, and segments were placed carefully on a 4-mm glass pipette. The serosal surface was scored with a dulled scalpel blade and the LMMP layer was gently peeled off with a wet cotton swab. The integrity of the LMMP was checked using light microscopy.
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Measurement of Acetylcholine Release Acetylcholine release was measured as described previously.3 Briefly, to obtain LMMP loaded with labeled acetylcholine, tissues were incubated with 0.2 mmol/L [methyl3 H]choline in Krebs’–Ringer’s solution containing the following (in mmol/L): NaCl, 120.9; KCl, 5.9; CaCl2 , 2.5; MgCl2 , 1.2; NaHCO3 , 15.5; NaH2PO4 , 1.2; and glucose, 11.1 for 1 hour at 377C and were oxygenated with 95% O2 and 5% CO2 . Tissues were washed subsequently twice in 5 mL of normal Krebs’ buffer and suspended in a water-jacketed superfusion chamber maintained at 377C using a thermostatically controlled water pump (Haake, Karlsruhe, Germany). The LMMP were superfused with oxygenated Krebs’ solution containing 10 mmol/L hemicholinium-3 at a flow rate of 1 mL/min using a peristaltic pump (Buchler, Fort Lee, NJ). Superfusate was collected at 2-minute intervals using an Altrorac 7000 fraction collector (LKB, Bromma, Sweden). After a 40-minute equilibration period, tritium release was stimulated by electrical field stimulation (EFS) and by a 50 mmol/L KCl-Krebs’ solution at 58 minutes. EFS was applied via two parallel silver electrodes at 10 Hz, 0.5 milliseconds, and 30 V for 1 minute. These parameters have been shown previously to optimally release [3H]acetylcholine from rat jejunal LMMP and to reflect stimulation of enteric neurons.3 After completing the protocol, 4 mL of aqueous scintillation fluid ACS (Amersham Corp., Oakville, Ontario, Canada) was added to each vial containing 2 mL of superfusate, and the tritium content was determined by a Beckman liquid scintillation counter (model LS 5801; Beckman Instruments, Inc., Fullerton, CA) with a counting efficiency of 35%. The tissues were blotted dry, weighed, and dissolved overnight in 1 mL of NCS-II tissue solubilizer. The solution was neutralized with 100 mL of glacial acetic acid. Four milliliters of scintillation fluid was added, and the tritium content was counted as described above to determine the remaining amount of radioactivity in the tissue. The evoked release of 3H on stimulation was expressed as the fractional release of the tritium present in the tissue at the time of stimulation as described previously.3 In an earlier study using high-performance liquid chromatography, we have shown that the evoked tritium release consists entirely of [3H]acetylcholine and therefore is an accurate reflection of functional neurotransmitter released during stimulation.3
Protocols Involving hr Cytokines To study immediate and delayed effects of hrLIF on H release, hrLIF at different concentrations (0.02–100 ng/ mL) was added directly to the superfusion buffer or to the preincubation media at various time points before onset of superfusion. Control tissues received the same volume of saline at equal time points. hrIL-1b was added to the incubation buffer 90 minutes before starting the superfusion at a concentration of 10 ng/mL. These parameters have been proven previously to result in a maximal suppression of acetylcholine release from rat jejunal LMMP.8 To study the specificity of hrLIF–mediated effects, the cytokine was incubated with an equal volume of 1 mg/mL goat polyclonal hrLIF-neutralizing 3
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antibody for 20 minutes at 377C. Twenty-milliliter aliquots of this mixture were added to the preincubation media for 90 minutes, resulting in a final hrLIF concentration of 20 ng/mL. Controls were treated with a mixture of sterile phosphatebuffered saline and hrLIF at an equal concentration.
Data Analysis All studies consisted of at least four separate experiments involving different animals. The data are expressed as mean { SE. For normally distributed data, Student’s t test was used to compare two groups, and one-way analysis of variance was used when comparing three or more groups. For nonparametric data, Mann–Whitney rank sum test and Kruskal–Wallis analysis of variance were applied, respectively. Statistical significance was inferred at P values of õ0.05.
Results hrLIF Effects on Acetylcholine Release To examine immediate effects of LIF on basal or stimulated acetylcholine release, hrLIF (final concentrations, 2 ng/mL and 20 ng/mL) was added directly to the superfusion solution during the equilibration period or 8 minutes before EFS or KCl stimulation. Addition of hrLIF directly to the superfusate solution did not cause any changes in basal or stimulated outflow of tritium from the LMMP (data not shown). Delayed effects of hrLIF on acetylcholine release were determined by incubating the tissues in oxygenated Krebs’ buffer at 377C 60 minutes before [methyl-3H]choline administration, resulting in a total incubation time of 120 minutes. hrLIF was added to the incubating LMMP at various time points. When added to the preincubation media 90 minutes before the onset of superfusion, hrLIF caused
Figure 2. Time course of hrLIF-induced suppression of 3H release. Tissues were preincubated for 120 minutes before superfusion. hrLIF (20 ng/mL) was added at indicated time points during the preincubation. 3H release was stimulated by EFS (h) or KCl (/). Data are expressed as the percentage of the stimulation observed after incubation with saline (control release). Values are expressed as mean { SE of four independent experiments. *Value significantly different from control (P õ 0.05).
a marked, dose-dependent suppression of acetylcholine release (Figure 1). A significant suppression of EFS-stimulated release occurred at hrLIF concentrations from 2 to 100 ng/mL (2 ng/mL: 52.0% { 16.8% of control fractional 3H release, P õ 0.05; 20 ng/mL: 23.4% { 9.7%, P õ 0.01; and 100 ng/mL, 39.4% { 13.3%, P õ 0.01). Similar results were obtained for KCl-evoked acetylcholine release (20 ng/mL: 32.3% { 6.0%, P õ 0.01; 100 ng/mL: 55.4% { 21.9% of control fractional 3 H release, P õ 0.05). The maximally effective concentration for LIF-induced suppression of acetylcholine release induced by either EFS or KCl was 20 ng/mL. As shown in Figure 2, a minimal incubation time of 60 minutes was required for a significant suppression of EFS-evoked acetylcholine release (36.2% { 0.15% of control fractional 3H release; P õ 0.01). Maximal suppression occurred after 90 minutes of preincubation (23.4% { 10.6% of control fractional 3H release; P õ 0.01). Impairment of KCl-evoked release only reached significance after 90 minutes of preincubation. Specificity of hrLIF Effects
Figure 1. Concentration dependence of hrLIF-induced suppression of 3 H release from rat LMMP preparations. Tissues were incubated for 90 minutes with saline or hrLIF in the concentrations indicated for 90 minutes before subsequent stimulation by EFS (h) or KCl (/). The release of acetylcholine was measured as 3H release and expressed as a percentage of the release obtained in the absence of LIF. Shown are mean { SE from four experiments. *Value significantly different from control (P õ 0.05).
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Using the optimal conditions for hrLIF suppression of 3H release (20 ng/mL and 90 minutes) as determined earlier, we repeated the cytokine incubation in a separate set of tissues. Specific hrLIF-neutralizing antibody (10 mg/mL) added to hrLIF before and during preincubation abolished the effects of hrLIF on stimulated 3 H release (Figure 3). Values for EFS- (1.53% { 0.45%) and KCl-evoked (1.01% { 0.17%) releases when incubated with hrLIF and antibody combined were not significantly different from controls (control EFS, 1.22% { 0.13%; KCl, 0.88% { 0.11% fractional 3H release), WBS-Gastro
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Figure 3. Effect of hrLIF-neutralizing antibody on LIF-induced suppression of EFS-evoked 3H release. LMMP preparations were incubated for 90 minutes with saline (h), hrLIF (20 ng/mL) ( ), hrLIF plus antibody (AB) (10 mg/mL) (j), or antibody alone ( ). Values are expressed as mean { SE from four experiments. *Value significantly different from control (P õ 0.001).
whereas hrLIF alone caused a significant suppression of the evoked 3H release (EFS: 0.22% { 0.07% fractional 3 H release, P õ 0.001; KCl: 0.30% { 0.06%, P õ 0.05). hrLIF-neutralizing antibody alone did not cause any significant changes in 3H release (Figure 3). Reversibility of hrLIF Effects To investigate the reversibility of the impaired H release by hrLIF, tissues were incubated with the cytokine (20 ng/mL) for 90 minutes, washed twice in normal Krebs’ buffer, and allowed to recover for 120 minutes in normal Krebs’ buffer before the onset of superfusion. As shown in Figure 4, the suppression of 3H release by hrLIF (20 ng/mL) was reversed by this washout procedure. EFS-stimulated release (hrLIF, 0.90% { 0.21%; vs. control, 0.89% { 0.19% fractional 3H release) and KCl-evoked release (hrLIF, 0.65% { 0.14%; vs. controls, 3
Figure 4. Reversibility of hrLIF effects on 3H release when stimulated by EFS (j) or KCl ( ). Tissues were incubated with saline or LIF (20 ng/mL), followed by a 60-minute incubation in normal Krebs’ buffer and 60-minute incubation with [3H]choline. Values are expressed as mean { SE of four independent experiments. cont, control.
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Figure 5. Effect of rat LIF-neutralizing antibody on IL-1b–induced suppression of (A ) EFS- and (B ) KCl-stimulated 3H release. Tissues were incubated with saline (h), IL-1b ( ), IL-1b plus antibody (AB) (j), or antibody alone ( ). Data are expressed as mean { SE from four experiments. *Significantly different from control (P õ 0.05).
0.49% { 0.09% fractional 3H release) were not significantly different in controls and hrLIF-incubated tissues. Interactions Between IL-1 and Endogenous LIF Incubation of LMMP with 10 ng/mL of hrIL-1 for 90 minutes resulted in a significant suppression of stimulated 3H release (EFS: IL-1b, 0.28% { 0.12%; vs. controls, 1.24% { 0.17%; P õ 0.01; KCl: IL-1b, 0.42% { 0.12%; vs. controls, 0.88% { 0.12% fractional 3H release; P õ 0.05) (Figure 5). Previous studies have shown that these conditions are optimal for IL-1–induced suppression of 3H release from myenteric plexus in rat jejunum.8 Addition of polyclonal anti-rat LIF antibody (20 mg/mL) to the incubation media abolished the effects of hrIL-1b on EFS- and KCl-evoked 3H release from the LMMP (Figure 5). EFS-evoked 3H release after incubation with IL-1b (10 ng/mL) and anti-rat LIF antibody was 1.02% { 0.09% (% fractional 3H release) vs. 1.24% { 0.17% in controls. Similarly the KCl-evoked release after exposure to IL-1 and anti-rat LIF antibody totaled 0.82% { 0.16% (fractional 3H release) vs. 0.87% { 0.11% in controls. Anti-rat LIF antibody alone did not cause any significant changes in 3H release. Addition WBS-Gastro
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Table 1. [3H]Choline Uptake of LMMP Preparations After Preincubation With hrLIF hrLIF (ng/mL)
0
0.2
2
20
100
[3H]choline uptake (1103 dpm/mg )
9.85 { 0.85
5.94 { 1.02
7.45 { 0.43
6.95 { 0.34
7.63 { 1.85
NOTE. hrLIF at different concentrations was added 90 minutes before onset of superfusion. [3H]choline uptake was calculated as the sum of the total 3H released during the experiment and the residual 3H in the tissue. Results were normalized for the weight of each LMMP. Values are expressed as mean { SE from four independent experiments. No significant differences (P ú 0.05) were observed between treatment groups using analysis of variance.
of anti-hrLIF antibody to the hrIL-1b preincubation did not interfere with IL-1b–induced suppression of 3H release (EFS, 0.31% { 0.07% vs. 1.24% { 0.17% control fractional 3H release; P õ 0.05; not significantly different from IL-1b alone). As an additional external control, IL1 incubation was repeated in the presence of normal rabbit serum (1:200 dilution). Addition of normal rabbit serum did not influence the effects of hrIL-1 on 3H release from LMMP (data not shown). Effects of hrLIF on 3H Content of the LMMP The total uptake of [methyl-3H]choline, as determined by the sum of 3H released from the LMMP and the residual 3H remaining in the tissue at the end of the experiment, per milligram of tissue weight was not significantly altered by hrLIF at different doses (Table 1). However, incubation of the tissue with IL-1b (10 ng/ mL) resulted in a significant suppression of [methyl3 H]choline uptake (IL-1b, 6.16 { 0.53 dpm/mg; control, 10.03 { 0.87 1 103 dpm/mg; P õ 0.01; n Å 5).
Discussion The results of the present study provide circumstantial evidence to support a role for the neuropoietic cytokine LIF as a downstream mediator of the suppressive effects of IL-1 on the myenteric plexus of the rat intestine. Previous studies in our laboratory have shown that the suppression of enteric neurotransmitter release by proinflammatory cytokines requires the induction of protein intermediates.8,9 However, no further data were obtained about the nature of these downstream mediators, and to the best of our knowledge, there are no reports available in the literature on secondary mediators of the actions of IL-1 on enteric cholinergic nerve function. The role of immune-regulatory cytokines in neuronal development and their response to injury has been characterized most extensively in autonomic ganglia of the rat.6,16 Normal neuronal maturation and injurious alterations are closely regulated by a variety of growth factors and cytokines. A key position in these neuroimmune interactions is occupied by an emerging family of cytokines that share several biological actions on neuronal and / 5e11$$0013
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nonneuronal cells through a common signal transduction pathway, the transmembrane glycoprotein gp130.17,18 From this group of cytokines, ciliary neurotrophic factor, cholinergic differentiation factor or LIF, IL-6, and IL-11, are thought to play an important role in neuroimmune interactions. LIF, originally discovered as a differentiation and inhibition factor of murine myeloma cell lines,19 has unique properties in various nerve types. It improves the maturation and survival of spinal motor neurons and sensory neurons from the dorsal root ganglia,20,21 it induces cholinergic phenotypic switch and neuropeptide expression in sympathetic nerve cultures,12 and it is involved in neuronal response to injury in vivo.22,23 In sympathetic ganglia, LIF has been shown to act as a soluble intermediate for the IL-1– and tumor necrosis factor a–induced expression of preprotachykinin messenger RNA and substance P.12,24 In the present study, we provide evidence that the cytokine cascade of LIF induction by IL-1 is probably not restricted to the sympathetic cervical ganglia but may be an important sequence in the observed injury-related alterations of intestinal nerve function. In our in vitro system, the action of LIF on cholinergic nerve function occurs at concentrations between 2 and 100 ng/mL (88 pmol/L to 4.4 nmol/L). Similar concentrations are required for the induction of substance P and choline-acetyl transferase, as well as for effects on cell survival in cultured sympathetic ganglia and somatic motor neurons.15,21,25,26 The concentrations of hrLIF required to suppress rat myenteric neurotransmitter release in the present study are also consistent with the reported affinity constant of the LIF receptor in murine cell lines (dissociation constant of 30–100 pmol/L).27 A different dose-response relation was observed for the impairment of KCl-induced 3H release by hrLIF compared with the EFS-evoked release from rat myenteric nerves. Earlier work in our laboratory has shown that different membrane-localized mechanisms are involved in KCl- and EFS-evoked 3H release in the rat LMMP superfusion system because the EFS-induced but not the KCl-induced release was totally inhibited by the axonal sodium channel inhibitor tetrodotoxin.3 Thus, differences in neurotransmitter release mechanisms triggered by EFS and WBS-Gastro
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KCL probably account for a different hrLIF sensitivity. The reversal of IL-1b suppression of acetylcholine release by specific anti-rat LIF antibody shows an interaction between these two cytokines in the myenteric plexus. The anti-rat LIF antibody used in this study was raised by immunizing rabbits with a synthetic peptide corresponding to the NH2 -terminal 11 amino acids of rat LIF; this was followed by purification of the antiserum based on its affinity for the synthetic peptide.15,28 This part of the native molecule is responsible for the bioactivity of LIF on sympathetic neurons.15 Previous studies have shown that this antibody lacks cross-reactivity with other neuropoietic cytokines, such as ciliary neurotrophic factor and IL-6.29 Furthermore, normal rabbit serum did not interfere with the IL-1–induced effects on neurotransmitter release in rat LMMP. Therefore, it is highly unlikely that the antibody neutralized the activity of a molecule other than LIF. In contrast, the lack of inhibition of hrIL-1b–mediated effects on rat intestinal LMMP by polyclonal anti–hrLIF most likely reflects differences between the amino acid sequences of human and rat LIF. The cascade of IL-1b induction of LIF as the downstream mediator for its effects on neurons was originally described in sympathetic ganglia and in bone marrow.13,30 We chose to investigate the effects of LIF on cholinergic myenteric nerve function because these neurons constitute a major intrinsic population in the myenteric plexus, and we have shown previously that IL-1– induced suppression of acetylcholine release from LMMP requires de novo protein synthesis. In contrast, previous studies from our group have shown that exogenous IL1b induces decreased norepinephrine release from rat myenteric plexus at least partially through the autoinduction of IL-1.9,31 The ability of LIF to suppress acetylcholine release in the present study does not reflect a reduction in the uptake of [3H-methyl]choline by the myenteric nerves in the LMMP. However, in myenteric plexus obtained from Trichinella spiralis–infected rat jejunum, suppression of acetylcholine release is accompanied by a reduced uptake of [3H-methyl]choline.3 Both alterations in myenteric nerve function were reproduced by exogenous hrIL-1b in normal rat LMMP in a previous study from our group,8 and this finding was confirmed in the present study. Therefore, it is unlikely that LIF accounts for all the changes in cholinergic nerve function attributed to IL-1 in the parasitized rat jejunum. In contrast to our results, comparable concentrations of LIF have been shown to increase acetylcholine content by up-regulating choline acetyltransferase in cultured sympathetic ganglia and motor neurons.15,21,32,33 However, these nerve cul/ 5e11$$0013
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tures were obtained from embryonic animals and did not show a cholinergic phenotype before exposure to LIF as opposed to the mature cholinergic enteric nerves present in the adult rat LMMP. Moreover, the induction of choline acetyltransferase in cultured embryonic nerves required several days of incubation with LIF compared with the 60 minutes in our system. The cellular source of LIF in the myenteric plexus is not clear. Various cell types are known to produce LIF in vitro when the appropriate stimulus is provided. Cultured fibroblasts and brain astroglia can be induced to secrete LIF,19,34 and rat peritoneal mast cells produce LIF in vitro when stimulated with a calcium ionophore.35 However, in normal rat LMMP, glial cells, macrophagelike cells, and fibroblasts are the most likely source of LIF because peritoneal mast cells are sparse in these preparations. Evidence implicating neuroglia as the source of LIF has been found in cultured sympathetic ganglia. In these mixed nerve cultures, IL-1–induced LIF secretion was eliminated when cultures were purified by antimitotic agents to eliminate nonneuronal foster cells.13,24 Furthermore, glial cells in culture are capable of producing LIF,13,34,36 and in situ studies have shown that peripheral nerve section induces increased LIF expression by Schwann cells.37 In the intestine, previous work in our laboratory has shown that hrIL-1b induces ultrastructural changes in macrophage-like cells of the myenteric plexus suggestive of phagocytosis and secretion.31 Nevertheless, we cannot exclude entirely a direct action of exogenous IL-1b on neuronal IL-1 receptors to induce LIF exerting an autocrine effect because neuronal IL-1 receptors have been described in the central nervous system.38 – 41 The functional relevance of cytokine-mediated effects on myenteric nerve function is supported by the recent report from our laboratory of copious in situ expression of IL-1b messenger RNA and increases in IL-1b protein in the jejunal LMMP region of nematode-infected rats.7 Contrary to IL-1b, constitutive LIF expression is not readily detectable in most tissues except for the pregnant uterus,42,43 but LIF appears to be up-regulated in inflammatory states. Elevated plasma levels of bioactive LIF have been reported in septic shock and synovial fluid of patients with arthritis has been found to contain significant amounts of LIF, similar to the concentrations that were required for the effects on enteric nerves in our study.44,45 In summary, we have shown that LIF impairs cholinergic nerve function in isolated rat myenteric plexus. Furthermore, the results from the present study provide evidence for the induction of LIF as a downstream mediator for the action of IL-1b on myenteric nerve function. Taken together, these findings extend the role of neuroWBS-Gastro
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poietic cytokines, such as LIF, as important modulators of the enteric nervous system. Further research will be required to establish the role of LIF in injury-related nerve dysfunction associated with experimental and clinical intestinal inflammation.
20.
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inhibitory factor levels are elevated in septic shock and various inflammatory body fluids. J Clin Invest 1992;90:2031–2037. 45. Warring PM, Carroll GJ, Kandiah DA, Buirski G, Metcalf D. Increased levels of leukemia inhibitory factor in synovial fluid from patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 1993;36:911–915.
Received February 6, 1996. Accepted May 24, 1996. Address requests for reprints to: Stephen M. Collins, M.D., Division of Gastroenterology, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. Fax: (905) 521-4958. Supported by a grant from the Medical Research Council of Canada (to S.M.C.) and by grants from the National Fonds voor Wetenschappelijk Onderzoek and D. Collen Research Foundation, Belgium (to G.V.A.). The authors thank Dr. P. H. Patterson (California Institute of Technology, Pasadena, CA) for his kind gift of purified rabbit anti-rat leukemia inhibitory factor antibody.
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