1 Department of Gastroenterology, University of Heidelberg, D-69115 Heidelberg, Germany; and 2 Intestinal Disease Research Program, McMaster University Medical Centre, Hamilton, Ontario, Canada L8N-3Z5
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ABSTRACT |
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As yet, little is known about the function of the glia of the
enteric nervous system (ENS), particularly in an immune-stimulated environment. This prompted us to study the potential of cultured enteroglial cells for cytokine synthesis and secretion. Jejunal myenteric plexus preparations from adult rats were enzymatically dissociated, and enteroglial cells were purified by complement-mediated cytolysis and grown in tissue culture. Cultured cells were stimulated with recombinant rat interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-
, and IL-6 mRNA expression and secretion were assessed using RT-PCR and a bioassay, respectively. Stimulation with TNF-
did
not affect IL-6 mRNA expression, whereas IL-1
stimulated IL-6 mRNA
and protein synthesis in a time- and concentration-dependent fashion.
In contrast, IL-6 significantly and dose-dependently suppressed IL-6
mRNA expression. In summary, we have presented evidence that enteric
glial cells are a potential source of IL-6 in the myenteric plexus and
that cytokine production by enteric glial cells can be regulated by
cytokines. These findings strongly support the contention that enteric
glial cells act as immunomodulatory cells in the enteric nervous system.
enteric glia; tissue culture
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INTRODUCTION |
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INFLAMMATORY CONDITIONS of the gut, ranging from peptic esophagitis to ulcerative colitis, induce alterations of gastrointestinal motility and sensory perception (for review, see Ref. 12). In animal models, a causal relationship between the presence of mucosal inflammation and altered sensory-motor function has been established (12). However, functional changes in the neuromuscular compartment of the gut wall can often be observed in the absence of an inflammatory infiltrate, which remains largely within the lamina propria (8, 12). Furthermore, neuromuscular dysfunction may persist after mucosal inflammation has subsided (5). These observations suggest that mediators of neuromuscular dysfunction are produced locally within the deeper neuromuscular layers of the intestinal wall.
Several studies have provided evidence that cytokines are important
mediators of neuromuscular dysfunction in the inflamed intestine
(13, 14, 28, 30, 39, 46, 50). Although activated
mononuclear cells are generally considered to be the primary source of
proinflammatory cytokines, findings suggesting that cytokine synthesis
in the myenteric plexus does not depend on the presence of immunocytes
implicate tissue structural cells as sources of cytokines in the
enteric nervous system (ENS) (12). This notion is
sustained by the recent finding that intestinal smooth muscle cells are
indeed sources of and targets for cytokines such as interleukin
(IL)-1 and IL-6, suggesting a role for these cells as a secondary
source of mediators in the amplification and perpetuation of the
inflammatory response (36, 37).
However, another cell type is in a strategically much better position to modulate nerve function in the neuromuscular compartment of the gut wall, namely the enteric glial cells (EGC). EGC are closest to the ganglionic neurons, comprising the interface between neurons and extraganglionic cells (17) like smooth muscle cells, fibroblasts, or resident macrophages, which have recently been described in the rat intestinal muscularis (35). Although EGC have been known for more than a century (reviewed in Ref. 49), little is known about their functional role in the ENS, particularly during intestinal inflammation.
This lack of knowledge is even more evident when the glial cells of the
ENS and the central nervous system (CNS) are compared. During CNS
inflammation cytokine synthesis is detectable long before mononuclear
cells have passed the blood-brain barrier, and there is evidence that
cytokine synthesis by resident CNS cells is essential for the
attraction of immunocytes and enables them to pass the blood-brain
barrier. So far, two different types of glial cells have been
demonstrated to produce cytokines in the CNS, namely, astrocytes
(15) and microglia (19, 20). The cytokine
whose synthesis has gained the highest attention is IL-6, and IL-6
production by CNS astrocytes has been demonstrated in response to a
vast array of stimuli, including IL-1, IL-6, tumor necrosis factor
(TNF)-
, and cAMP (6, 7, 22, 23, 51, 52, 55).
In view of the potential of CNS astrocytes to synthesize IL-6, we designed the present study to investigate whether this capacity is shared by glial fibrillary acidic protein (GFAP)-positive EGC. Because the understanding of the physiological properties of specific cell types is greatly facilitated by the ability to study purified populations of these cells in vitro, we have developed a procedure that allows us to culture EGC without contamination by other cell types (45, 47). Using this approach, we have found evidence that EGC are a potential source of cytokines in the myenteric plexus and that cytokine production by EGC can be regulated by cytokines. These findings strongly support the contention that EGC play a key role in the immunomodulation of enteric nerve function.
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MATERIALS AND METHODS |
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Materials
DMEM, FBS, and horse serum were purchased from Life Technologies Gibco (Karlsruhe, Germany); Hanks' balanced salt solution (HBSS), HEPES, antibiotic-antimycotic solution, trypsin, cytosine arabinoside (Ara-C), forskolin, Triton X-100, bovine serum albumin (BSA), 3,3'-diaminobenzidine tetrahydrochloride (DAB), and peroxidase-antiperoxidase complex (PAP) from Sigma-Aldrich (Deisenhofen, Germany); and bovine pituitary extract (BPE) from Linaris (Wertheim, Germany). Polyclonal rabbit anti-cow antibodies directed against GFAP and S-100 and horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig were supplied by Dako (Hamburg, Germany). Dispase was from Boehringer Mannheim Biochemica, and murine monoclonal anti-CD 90 (mouse anti-rat Thy-1.1; MRC OX-7), HRP-conjugated goat anti-mouse Ig, and preimmune sera used for controls were purchased from Camon-Serotec (Wiesbaden, Germany). Guinea pig complement was obtained from Biozol (Eching, Germany). Rat recombinant (rr) IL-1Preparation of EGC Cultures
Primary cultures of enteric glia were generated from enzymatically dissociated preparations of rat longitudinal muscle-myenteric plexus preparations as previously described (47). Briefly, the entire jejunum was taken from adult male Sprague-Dawley rats (180-200 g) under sterile conditions and divided into segments ~4-6 cm in length that were opened along the mesentery and pinned flat with the serosal side up. Under a Leitz dissection microscope, the myenteric plexus with the longitudinal muscle layer attached was gently peeled off the underlying circular muscle using a pair of fine dissection forceps, cut into pieces of ~5 mm, and placed into 6 U/ml dispase in DMEM at 37°C for 30-60 min. Dissociated cells were plated into T-75 flasks and maintained in DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic solution, and the antimitotic agent Ara-C at a concentration of 10Immunocytochemistry
Cultured cells were morphologically and immunocytochemically characterized. Morphological assessment was performed by phase-contrast microscopy. To further identify EGC and to exclude contamination with other cell types, tissue cultures were stained with polyclonal antibodies directed against GFAP and S-100, respectively, and a monoclonal antibody directed against Thy-1.1. GFAP is a cytoskeletal intermediate filament protein, whereas S-100 is an intracellular acidic protein that binds Ca2+ and belongs to a family of calcium-binding proteins (11). In the gastrointestinal tract, both GFAP and S-100 are considered to be specific markers of glial cells (16, 31). Thy-1.1 is an epitope present on the surface of rodent myofibroblasts (2, 9). For immunohistochemistry, EGC were plated onto 22-mm2 glass coverslips, placed into tissue culture dishes, and grown to subconfluence. All immunostaining procedures were performed at room temperature.For GFAP and Thy-1.1 immunolabeling, cultures were fixed in acid
ethanol and blocked with 5% horse serum in PBS for 30 min. Subsequently, they were incubated with rabbit anti-GFAP antibody (1:200
dilution) or murine monoclonal anti-CD90 antibody (MRC OX-7; 1:100
dilution) for 60 min, thoroughly washed in PBS, and incubated with
HRP-conjugated goat anti-rabbit Ig (1:500 dilution) or HRP-conjugated
goat anti-mouse Ig (1:500 dilution), respectively, for a further 60 min. After another washing step, PAP (1:150 dilution) was added for 30 min. Finally, DAB (1 mg/ml) made up freshly in PBS with 0.01%
H2O2 was added to the rinsed coverslips for
3-15 min, during which time color development was visually
controlled. For S-100 immunolabeling, cultures were fixed in 4%
paraformaldehyde for 10 min followed by treatment with methanol at
20°C for 10 min. The subsequent steps were identical to the GFAP
immunolabeling protocol, except that anti-S-100 antibody (1:200
dilution) was used as primary antibody. Negative controls using
preimmune rabbit or mouse serum, respectively, and the relevant
secondary antibodies were included in all experiments. A nuclear
hematoxylin counterstain was added after immunolabeling if
required. After completion of the staining procedures, coverslips
were mounted on glass slides and examined with ×10, ×20, or
×40 objectives on a Zeiss microscope.
Assessment of IL-6 Production by EGC
Stimulation protocols.
To investigate whether EGC can synthesize IL-6, EGC were plated into
T-75 flasks and grown to subconfluence. Growth medium was changed twice
weekly and 24 h before the experiments. At the beginning of each
experiment, medium was removed and cultures were carefully washed. DMEM
supplemented with 10% FBS and the required concentration of rrIL-1,
rrTNF-
, or rrIL-6 was then added to the cells. Control cells were
incubated with DMEM plus 10% FBS. A series of control experiments was
performed in which the cytokines were heat inactivated by vigorous
boiling for 20 min.
RNA extraction and IL-6 RT-PCR. After stimulation of the cells, total cellular RNA was harvested using a kit based on the phenol-chloroform extraction procedure followed by RNA precipitation with isopropanol (Fast RNA Green Kit; Oncor Appligene, Heidelberg, Germany). The yield of RNA was quantitated spectrophotometrically by absorption at 260 nm. The quality of RNA was checked by formaldehyde agarose gel electrophoresis.
Synthesis of full-length cDNA templates for RT-PCR was performed at 37°C for 60 min after RNA samples had been denatured at 65°C for 10 min. For reverse transcription we used glassified reaction mixes made up from Not I-(dT)18 primer, M-MuLV reverse transcriptase, RNase inhibitor, and nucleotides (Ready-To-Go T-Primed First-Strand Kit; Amersham Pharmacia Biotech, Freiburg, Germany). Semiquantitative PCR amplification of the resulting cDNA was performed using specific primers (10 pmol/µl) for rat IL-6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene and a master mix consisting of Taq DNA polymerase and nucleotides in the appropriate buffer system. The master mix was provided in an ambient-temperature-stable reaction bead (Ready-To-Go PCR Beads; Amersham Pharmacia Biotech). The sequence of the forward primer used for IL-6 amplification was 5'GAGGATACCACCCACACCAGACCAGTA 3' (172 bp-198 bp) and of the reverse primer was 5'GGTTTGCCGAGTAGACCTCATAGTGAC 3' (670 bp-696 bp) resulting in a PCR product of 525 bp. IL-6 amplification was performed for 35 cycles with an annealing temperature of 66.5°C. The sequence of the forward primer for GAPDH amplification was 5'ACTGGCGTCTTCACCACCAT (319 bp-338bp) and of the reverse primer 3'TCCACCACCGTGTTGCTGTA (982 bp-1,001 bp), resulting in a PCR product of 683 bp. GAPDH amplification was performed for 26 cycles with an annealing temperature of 68°C. Identical amounts of cDNA were used for IL-6 and the corresponding GAPDH amplifications. Controls using RNA samples without reverse transcription or controls without cDNA were used to demonstrate absence of contaminating DNA. PCR products were analyzed on ethidium bromide-stained 1.5% agarose gels, and densitometry was performed using a Bioprofil 1D image analysis system with Bio-1D V.97 software (Vilbert Lourmat, France).Measurement of IL-6 protein.
After cell stimulation, culture media were sterilely removed and spun
at 3,500 rpm for 20 min to obtain cell-free supernatants. These
supernatants were frozen and kept at 70°C for further processing but never for more than 2 wk.
Data Expression and Statistical Analysis
After PCR amplification, signal intensity of IL-6 and GAPDH PCR products was quantified densitometrically and relative amounts of IL-6 amplimers were normalized to the values for GAPDH amplimers for each experiment. Ratios of IL-6 amplimers to GAPDH amplimers were expressed as a percentage of control. Each experiment was repeated six times using different cell culture preparations, and the resulting ratios are summarized as means ± SE. Differences between the different experimental groups were analyzed by one-way ANOVA followed by post hoc Scheffé tests.Variances of IL-6 protein data were stabilized using logarithmic transformation. Transformed data were analyzed by Kruskal-Wallis ANOVA, and post hoc comparisons between groups were done by Mann-Whitney U-test for independent samples. In all analyses, the level of statistical significance was set at P < 0.05.
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RESULTS |
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Morphology and Growth Pattern of Purified EGC In Vitro
Purified cultures of EGC consisted of homogeneous cell populations as evidenced by phase-contrast microscopy and immunohistochemistry (Fig. 1). When subconfluent, EGC typically exhibit a spindle-shaped or stellate morphology. Proliferation continues until stable flat monolayers are formed. Confluent cells are polygonal with indistinct or invisible intercellular borders. Immunohistochemical analyses confirmed that purified cultures were free from contaminating cell types. Although 100% of the cells exhibited strong immunoreactivities for the enteroglial markers GFAP and S-100 (Fig. 1), no immunoreactivity was detectable for the fibroblast marker Thy-1.1.
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IL-6 Production by Cultured EGC
Induction of IL-6 mRNA expression.
To assess whether IL-6 transcription can be induced in EGC,
subconfluent cells were incubated with rrIL-1 or rrTNF-
at
concentrations ranging from 1 to 100 ng/ml for 2, 4, 8, 12, 24 and
48 h. As shown in Fig. 2, there was
a minute amount of constitutive IL-6 mRNA expression. Stimulation of
EGC with TNF-
did not affect IL-6 mRNA expression (data not shown).
In contrast, when EGC were stimulated with IL-1
, a marked
upregulation of IL-6 transcription occurred that was time- and
concentration dependent (Fig. 2). The maximum increase of IL-6 mRNA
expression occurred after 24 h of stimulation with 100 ng/ml of
IL-1
(Fig. 2E). Densitometric analysis of IL-6 mRNA
expression after 24 h was performed for six separate experiments and revealed a significant increase after stimulation with IL-1
at
concentrations of 10 (175 ± 32% of control; P < 0.02) and 100 (280 ± 65% of control; P < 0.01)
ng/ml (Fig. 3).
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Effect of IL-1 on IL-6 protein.
To examine whether the observed effects of IL-1
on IL-6 mRNA
expression translated into IL-6 protein synthesis and secretion, purified EGC were incubated with rrIL-1
at a concentration of 100 ng/ml for 2, 4, 8, 12, and 24 h. After cell stimulation, IL-6 bioactivity in tissue culture supernatants was assessed using the B9
assay. In supernatants from nonstimulated controls, a very small amount
of IL-6 bioactivity was detectable, probably reflecting basal secretion
of IL-6 (0.33 ± 1.1 ng/ml). After 12 h of stimulation detectable IL-6 bioactivity was raised to 29.2 ± 12.6 ng/ml
(P < 0.01 compared with control), and after 24 h
IL-6 bioactivity in the supernatants was 107.7 ± 32.6 ng/ml
(P < 0.01) (Fig. 4).
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Specificity of action of IL-1.
Another set of experiments was performed to investigate whether the
observed effects of IL-1
on IL-6 production were receptor mediated.
Preincubation of EGC with a specific IRAP at a concentration of 10 µg/ml for 30 min completely blocked the effects of subsequent stimulation with IL-1
(10 ng/ml) (110 ± 36% of control),
indicating that these effects are receptor mediated (Fig.
5). IRAP alone did not significantly
affect IL-6 transcription (80 ± 42% of control; Fig. 5).
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Autoregulation of IL-6 expression.
In contrast to the upregulation of IL-6 mRNA expression that occurred
after stimulation of EGC with exogenous IL-1, there was a
dose-dependent inhibition of IL-6 expression when cells were incubated
with exogenous IL-6 for 24 h (Fig.
7). IL-6 significantly suppressed IL-6
mRNA expression in concentrations as low as 1 ng/ml (80 ± 2.5%
of control; P < 0.05). This effect was more pronounced with higher cytokine concentrations, reaching a maximum after stimulation with 100 ng/ml of IL-6, which suppressed IL-6 mRNA expression to 57 ± 13% of control (P < 0.01;
Fig. 7).
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DISCUSSION |
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We present evidence here that EGC are a potential source of
cytokines in the myenteric plexus and that cytokine production by EGC
can be regulated by cytokines. These findings strongly support the
contention that EGC play a key role in the immunomodulation of enteric
nerve function. Specifically, we have demonstrated that EGC from rat
myenteric plexus produce the proinflammatory cytokine IL-6 and that
this is regulated by the proinflammatory cytokines IL- and IL-6 but
not TNF-
.
To assess the ability of EGC to produce cytokines, we have used a novel tissue culture technique yielding purified cultures of EGC from the myenteric plexus of adult rat intestine (45, 47). We have chosen to study the immunophysiological properties of EGC from adult tissues because this approach may offer several advantages over embryonic or newborn tissues. Because the effect of development in an artificial environment should be minimized in such cultures, findings in cultures derived from mature intestine may be more directly comparable with adult tissue (48). This implies a clear superiority of such cultures for our purposes because the investigation of enteroglial immunophysiology is relevant for inflammatory bowel disease (IBD), which occurs mostly in adolescent or adult intestine. Furthermore, adult tissues tend to be more readily available and are more easily handled experimentally.
EGC cultures were purified by a combination of the antimitotic agent Ara-C and complement-mediated cell lysis (47). This procedure provides tissue cultures consisting of homogeneous populations of distinctive stellate or spindle-shaped cells that were morphologically assessed as EGC. Lineage and purity of the cells were further ascertained by immunohistochemistry using antibodies directed against GFAP and S-100, which in the gastrointestinal tract are considered to be specific markers of glial cells (16, 31). Cultures containing typical EGC as predicted by morphology showed 100% positive staining with both antibodies, supporting the notion that we have raised pure EGC populations.
This purity allows for the first time the use of EGC for experimentation with subsequent molecular and biochemical analyses of cell products and hence offers the opportunity to study the role of EGC in the inflammatory response of the neuromuscular compartment of the gut wall. A recent study in transgenic mice found that ablation of enteric glia is followed by fulminant jejunoileitis, implicating these cells in the maintenance of small bowel structure and function (10). Nevertheless, the immunophysiological properties of enteric glia and their contribution to the development and propagation of IBD are not well understood. Enteric glia resembles central nervous system astroglia in its anatomic relationships to neurons and shares the expression of a number of antigens with astrocytes and non-myelin-forming Schwann cells of the peripheral nervous system (16, 31-33). These anatomic and molecular similarities suggest that enteric glia, astrocytes, and nonmyelinating Schwann cells may share common functions. Astrocytes both produce and respond to a variety of cytokines and have long been implicated in an array of immunological processes within the CNS (42). However, because there are fundamental differences between the CNS and ENS in terms of anatomic composition and overall function, cellular functions may be significantly different between CNS and ENS glia despite morphological similarities. Compared with published data on astrocytes, our findings in EGC suggest both functional similarities and significant differences between astrocytes and EGC.
Studying IL-6 expression in isolated and purified EGC, we have found
that stimulation of these cells with TNF- did not affect IL-6
transcription, whereas stimulation with IL-1
resulted in a marked
time- and concentration-dependent upregulation with a maximum increase
after 24 h of stimulation. Assessment of IL-6 bioactivity in
tissue culture supernatants confirmed that augmented IL-6 mRNA
expression is translated into IL-6 protein synthesis and secretion with
corresponding time courses.
In the CNS, astrocytes synthesize IL-6 in response to stimulation with
IL-1 but, in contrast to enteric glia, also in response to TNF-
(7, 43, 52). Moreover, IL-6 stimulates its own expression
in astrocytes (53), whereas we have found a significant and dose-dependent inhibition of IL-6 expression when cells were incubated with exogenous IL-6, providing evidence for a feedback inhibition of IL-6 production in enteric glia. This feedback inhibition may play a crucial role in the regulation of inflammatory processes in
the ENS and may provide a functional basis for the postulated anti-inflammatory potential of EGC (10).
In astrocytes, IL-1 effects are receptor mediated (4,
25). Similarly, our data provide functional evidence for IL-1 receptors on EGC because IL-1
-induced IL-6 expression could be inhibited by a specific IL-1 receptor antagonist. The heat sensitivity of the recombinant cytokine effects proves that the actions of IL-1
and IL-6 are not attributable to endotoxin or other heat-resistant bacterial contaminants.
The physiological consequences of IL-6 production by EGC are as yet unknown. In the CNS, the function of this cytokine is complex; IL-6 exerts neurotrophic and neuroprotective effects (23, 52), but it can also function as a mediator of inflammation, demyelination, and astrogliosis, depending on the cellular context (23, 40, 51, 52, 55). Studies in IL-6-deficient mice have demonstrated that IL-6 is crucial for the recruitment of inflammatory cells and activation of glial cells after brain injury with disruption of the blood brain barrier (44). However, in the same model it was demonstrated that IL-6 is important for neuroprotection and may facilitate nerve regeneration (44).
In the intestine, proinflammatory cytokines have been demonstrated to play a key role in the pathogenesis of IBD. Specifically, a role for IL-6 in the pathogenesis of Crohn's disease has been repeatedly demonstrated (21, 29). Elevation of local IL-6 activity may be a characteristic feature of active IBD, and macrophages and colonic epithelial cells have been implicated as the major cell types responsible for this phenomenon in the mucosa of patients with chronic intestinal inflammation (27, 38). Recently, a crucial role for IL-6 in the pathogenesis of murine colitis has been suggested, and anti-IL-6 receptor monoclonal antibody therapy has been proposed for treatment of human Crohn's disease (56). In another recent report, the mechanisms by which IL-6 may contribute to the perpetuation of chronic intestinal inflammation have been further elucidated because it was demonstrated that IL-6 mediates resistance of T cells to apoptosis in Crohn's disease (3).
On the basis of these observations, we propose that enteric glia is involved in the immune response in the neuromuscular compartment of the gut wall by production of IL-6. It has been demonstrated that IL-6 levels in the neuromuscular layers of the uninflamed intestine wall are low (37), whereas elevated expression occurs in infection and inflammation (12, 37). The observation that there is constitutive expression of inflammatory cytokines in the absence of an inflammatory infiltrate argues for the existence of a tissue-resident source of cytokines. Furthermore, in most animal models of intestinal inflammation, the inflammatory infiltrate originates in the mucosa and lamina propria and does not penetrate the myenteric plexus. Nevertheless, intestinal inflammation is accompanied by marked neuromuscular dysfunction (12, 54). Because it has been demonstrated that cytokines alter myenteric neural function (14, 28, 46), inflammation-associated neuromuscular dysfunction can be assumed to reflect a cascade of events involving the induction and release of cytokines within the plexus. The question emerges as to the origin of these cytokines in the myenteric plexus. Although other cell types have also been proposed to play a role in immune processes in the neuromuscular layers of the gut wall and to modify the immunological environment (8, 26, 36), EGC could nevertheless be the most strategic cell type for the amplification and perpetuation of the inflammatory response in the ENS because of their close anatomic association with enteric neurons and their location at the edge of the plexus ganglia, which provide them with features of a putative interface between the various extraganglionic cells and intraganglionic neurons (18).
In summary, we have provided evidence that EGC produce the proinflammatory cytokine IL-6 using purified primary cultures of EGC from adult rat myenteric plexus. On the basis of these findings, we propose that activated enteric glia may be the predominant source of IL-6 in the ENS, contributing to the amplification and perpetuation of the inflammatory response of the neuromuscular compartment of the gut wall.
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ACKNOWLEDGEMENTS |
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We thank Jane Anne Schroeder for expert technical help with the B9 bioassay and Dr. J. Gauldie, Dept. of Pathology, McMaster University, for the B9 hybridoma cells.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (Ru 528/5).
Address for reprint requests and other correspondence: A. Rühl, Innere Medizin IV, Universitätsklinikum, Bergheimer Str. 58, D-69115 Heidelberg (E-mail: anne.ruehl{at}med.uni-heidelberg.de).
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.
Received 5 November 2000; accepted in final form 22 January 2001.
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