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Am J Physiol Gastrointest Liver Physiol 293: G250-G255, 2007. First published April 12, 2007; doi:10.1152/ajpgi.00545.2006
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INFLAMMATION/IMMUNITY/MEDIATORS

Cytokine modulation of muscarinic receptors in the murine intestine

Hirotada Akiho,1,2 Waliul I. Khan,1 Atheer Al-Kaabi,1 Patricia Blennerhassett,1 Yikang Deng,1 and Stephen M Collins1

1Intestinal Disease Research Program, Department of Medicine, McMaster University, Hamilton, Ontario, Canada; 2and Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Submitted 26 November 2006 ; accepted in final form 5 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The extent to which gut motility and smooth muscle contractility are altered by intestinal inflammation depends on the nature of the underlying immune activation. The muscarinic receptor on smooth muscle plays a critical role in mediating acetylcholine-driven motor function. We examined the ability of cytokines to influence muscarinic receptor characteristics on intestinal longitudinal muscle and related the findings to studies on carbachol-induced contraction. Cells were isolated from longitudinal muscle myenteric plexus (LMMP). Cytokine receptor expression, muscle contractility, and muscarinic agonist receptor characteristics were examined by agonist displacement of [N-methyl-3H]scopolamine ([3H]NMS) binding. The TGF-beta1 receptor (543 bp) and the IFN-{gamma} receptor 1 (660 bp) were identified on smooth muscle cells. Scatchard analysis revealed dissociation constant and maximum binding values for [3H]NMS of 2.6 nM and 2.4 x 104 sites/cell, respectively, in control cells. Nematode infection was accompanied by a reduction in inhibitory constant of the high-affinity sites (KH), and this was independent of signal transduction and activator of transcription 6. Preincubation with TGF-beta1 enhanced longitudinal muscle contractility and decreased the KH to 2.2 pM (increased muscarinic receptor affinity), whereas preincubation with IFN-{gamma} increased the KH to 0.4 µM (decreased muscarinic receptor affinity) and decreased longitudinal muscle contractility. Preincubation of LMMP with IL-13 decreased the KH to 0.2 nM. Cytokines exert differential effects on the muscarinic receptor on intestinal longitudinal smooth muscle. These findings explain the basis for altered muscle contractility observed in Th1 and Th2 models of inflammation, as well as in the post-nematode-infected state.

interleukin-13; transforming growth factor-beta; intestinal motility

INFLAMMATION AND IMMUNE ACTIVATION in the gastrointestinal tract lead to altered motor function that can sometimes persist after resolution of the mucosal inflammatory response. Changes in motor function have been described in experimental models following a variety of inflammatory stimuli, including infection (5, 32), chemical irritation (23), and immune activation (25). In the context of infection, changes in motor function have been linked to host defense by enhancing the expulsion of the infectious agent. From a clinical viewpoint, some motility disorders have been associated with evidence of immune activation, and these range from mild (8) to moderately severe cases (30) of irritable bowel syndrome (IBS) to cases of life-threatening intestinal pseudo-obstruction (11). An understanding of the mechanisms underlying immune-mediated changes in gut motor function is therefore critical, not only in understanding the pathophysiology of but also in devising new therapeutic strategies for these disorders.

Because nematode infection generates a strong Th2 response in most hosts, studies have focused on the role of Th2 cytokines in mediating the hypercontractility of intestinal longitudinal muscle to carbachol stimulation, a hallmark of this model. Results indicate that hypercontractility is mediated by IL-4 and IL-13 acting via a signal transduction and activator of transcription (STAT) 6-dependent mechanisms (1, 19). In models where hypercontractility persists after infection, TGF-beta has been implicated as a mediator of the long-term changes in muscle function (2). However, recent observations in mice that are vulnerable to nematode infection indicate that changes in muscle contractility are quite different from those observed in strong Th2-responding hosts (22). Specifically, AKR mice infected with Trichuris muris are unable to expel the parasite and generate a Th1 immune response, resulting in hypocontractility of gut muscle (22). Similarly, when the Th1-promoting cytokine IL-12 is overexpressed in NIH Swiss mice before nematode infection (18), longitudinal muscle hypercontractility is markedly attenuated and worm expulsion is delayed. Together, these observations suggest that Th1 and Th2 immune responses exert opposing effects on longitudinal muscle contractility in the gut. Because each of these studies used carbachol as the contractile stimulus, these observations also suggest that cytokine modulation of the muscarinic receptor is pivotal in determining the nature of altered longitudinal muscle contractility in the gut.

Although the fundamental role of muscarinic receptors in the control of intestinal motility is generally acknowledged, it is now evident that this receptor plays a critical role in immune-mediated pathologies outside the gut. For example, it is now believed that immune activation suppresses inhibitory muscarinic M2 receptors in the airway, resulting in more acetylcholine release and M3-mediated bronchoconstriction (17).

In this study, we first determined the expression of cytokine receptors on longitudinal smooth muscle and then examined their ability to modify both the ligand-recognition properties of the muscarinic receptor on intestinal longitudinal muscle cells and the corresponding changes in longitudinal muscle contractility. We show that the muscarinic receptor is significantly influenced by the cytokines IFN-{gamma} and TGF-beta and that these cytokines have opposing effects on longitudinal muscle contractility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The following materials were used in this study: collagenase (CLS type 1), trypsin inhibitor, BSA, carbamylcholine chloride (carbachol), and atropine from Sigma (St. Louis, MO); HEPES from Bioshop Canada (Burlington, ON, Canada); IL-4, IL-13, IFN-{gamma}, and TGF-beta1 from R&D Systems (Minneapolis, MN); DMEM and antibiotic-antimycotic from GIBCO-BRL Life Technologies (Gaithersburg, MD); [N-methyl-3H]scopolamine([3H]NMS) from PerkinElmer Life Sciences (Boston, MA); aqueous counting scintillant (ACS) and NCS-II from Amersham Canada (Oakville, ON); and RNeasy mini kit from Qiagen (Mississauga, ON).

Mice. Studies were performed on male C57Bl/6 mice with or without Trichinella spiralis infection and STAT6–/– mice between 6 and 10 wk of age. STAT6–/– mice on a C57Bl/6 background were originally produced by gene mutation as described by Takeda et al. (29). Breeding pairs of STAT6–/– mice and their wild-type littermates (STAT6+/+) were obtained from the John Curtin School of Medical Research (Australian National University, Canberra, Australian Capital Territory, Australia). Mice were kept in filter-isolated cages in groups of four to five in positive-pressure rooms with a constant ambient temperature and a 14:10-h light/dark cycle. All experiments were approved by the Animal Care Committee at McMaster University and were conducted in accordance with the guidelines of the Canadian Council on Animal Care.

Trichinella infection. Mice were infected by the administration of 0.1 ml of phosphate-buffered saline containing 375 T. spiralis larvae by gavage. The larvae were obtained from infected rodents 60–90 days after infection by using a modification of the technique described by Castro and Fairbairn (7). The T. spiralis culture originated in the Department of Zoology at the University of Toronto, and the colony was maintained through serial infections alternating between male Sprague-Dawley rats and male CD1 mice.

Preparation of dispersed smooth muscle cells. Muscle cells were isolated from the longitudinal muscle myenteric plexus (LMMP) of the C57Bl/6 mice jejunum by a method similar to that used by Bitar and Makhlouf (6) to prepare smooth muscle cells from the guinea pig stomach. The uninfected mice and the mice infected with T. spiralis were killed by cervical dislocation. The jejunum was removed and placed in DMEM with 1% antibiotic-antimycotic. LMMP was peeled off from jejunum. The LMMP were preincubated with or without cytokines (10 ng/ml of IL-4, IL-13, TGF-beta1, or IFN-{gamma}) for 16 h in the 5% CO2 incubator. The LMMP was incubated for two successive 10-min periods at 31°C in 10 ml of HEPES medium (in mM: 98.1 NaCl, 3.87 KCl, 2.42 NaH2PO4·H2O, 4.86 L-glutamic acid, 4.86 fumaric acid, 4.86 pyruvate, 11.17 glucose, 1.79 CaCl2, 1.2 MgSO4·7H2O, and 23.5 HEPES, pH 7.4) containing 1 mg/ml of collagenase, BSA, and trypsin inhibitor. After incubation, the partly digested LMMP were washed with enzyme-free HEPES medium and were reincubated in 10 ml of fresh HEPES medium to allow the cells to disperse spontaneously. Cells were then harvested by filtration through a 210-µm polyester mesh.

Detection of TGF-beta1 in muscle layer and of IFN-{gamma} receptor 1 and TGF-beta type II receptor in muscle cells by RT-PCR. Expression of mRNA for TGF-beta1 in LMMP and mRNA for IFN-{gamma} receptor 1 and TGF-beta type II receptor in dispersed longitudinal single smooth muscle cells from control mice was investigated by a method described previously (31). Total cellular RNA was isolated by using the RNeasy mini kit according to the manufacturer's directions. The concentration of RNA was determined by measuring absorbance at 260 nm, and its purity was confirmed by using the ratio of absorbency at 260 nm to that at 280 nm. RNA was stored at –70°C until it was used for RT-PCR. mRNA was then reverse transcribed as described previously to yield cDNA, and the cDNA was amplified by PCR by using gene-specific primers. cDNA (0.1 µg) in 50-µl aliquots were then mixed with 20 pM of upstream and downstream primers, which were designed based on the available cDNA sequence. Primers were as follows: TGF-beta1, upstream 5'-TCA CCC GCG TGC CTA ATG GT and downstream 5'-GGA GCT GAA GCA ATA GTT GG-3' (10); IFN-{gamma} receptor 1, upstream 5'-GAC TGA TTC CTG CAC CAA CAT T-3' and downstream 5'-TTT ACC ACA GAG AGC AAG GAC T-3' (34); TGF-beta type II receptor, upstream 5'-TGT GGA CGC GCA TCG CCA GC-3' and downstream 5'-ACA CGG TAG CAG TAG AAG AT-3' (28). PCR was performed in 50-µl volumes containing 200 µM dNTP, 1.5 mM MgCl2, and 2.5 U Taq polymerase with corresponding buffer and distilled water. Messages for TGF-beta1 were coamplified for 39 cycles by using the following parameters: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s. Messages for IFN-{gamma} receptor 1 and TGF-beta type II receptor were coamplified for 34 cycles by using the following parameters: denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s. PCR products of 543 bp (TGF-beta1), 660 bp (IFN-{gamma} receptor 1), 526 bp (mTGF-beta receptor II1), and 601 bp (mTGF-beta receptor II2) were separated by 2.5% agarose gel electrophoresis and then were visualized under ultraviolet light after ethidium bromide staining. The ratio of TGF-beta1 gene expression compared with GAPDH expression was calculated.

[3H]NMS binding assay. In the radioligand-binding assay, muscle cells were suspended in HEPES medium containing 1% BSA. Duplicate aliquots (320 µl) of cell suspension containing 5 x 105 cells were incubated for 15 min at room temperature with 0.1–10 nM [3H]NMS alone or in the presence of 10 µM atropine or 10–3–10–12 M carbachol. The final incubation volume was 400 µl. The tubes were spun at 15,000 g for 2 min. The supernatant was removed by vacuum aspiration, and the pellet was washed with iced buffer three times. The tip of the Microfuge tube containing the pellet was severed, placed in 1 ml of tissue solubilizer (NCS-II), and allowed to dissolve overnight before the addition of 5 ml ACS and the counting of radioactivity in a Beckman LS5801 liquid scintillation counter at 34–37% efficiency. Nonspecific binding was measured as the amount of radioactivity associated with the muscle cells in the presence of 10 µM atropine. Specific binding was calculated as the difference between total and nonspecific binding (90 ± 3%). The Scatchard binding data and competition curve were analyzed by using PRISM software (GraphPad). Values for dissociation constant (KD) and maximum binding (Bmax) were calculated from Scatchard binding data. The concentration of radiolabeled ligand that produces 50% of the maximum occupancy is the KD, which associated with the receptor to deduce ligand affinity. Bmax is the top of the saturation plot curve, which is approximately equal to the number of binding sites. Inhibitory constant (Ki) values were calculated from IC50 (concentrations of competitor that reduced specific binding by 50%). KH and KL represent the Ki of agonist calculated from the high-affinity and low-affinity component of [3H]NMS binding, respectively.

Measurement of contraction and relaxation in dispersed cells. Dispersed cells were stimulated by the addition of a 0.8-ml aliquot of the cell suspension to 0.1 ml of the test agent and then incubated at room temperature for 30 s, because we previously found that carbachol induced the maximal contractile response in jejunal longitudinal smooth muscle cells after 30 s of incubation. The reaction was interrupted by the addition of acrolein in a final concentration of 1%. The median cell length of 30 cells on each slide was measured with a microscope using image-splitting micrometry, and the percent decrease from control in the mean cell length was determined.

Statistics. Each experiment was performed at least four times, and results are presented as means ± SE. Statistical analyses were performed by using the Student's t-test for comparison of two means or one-way ANOVA for the comparison of more than two means. A P value <0.05 was considered to be statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The expression of cytokine receptors on longitudinal smooth muscle cells. We have previously (1) identified the IL-4 receptor {alpha}-chain on murine intestinal muscle cells. We next determined whether receptors for the cytokines INF-{gamma} and TGF-beta1 are expressed on these cells. As shown in Fig. 1, the mRNA expression for TGF-beta1 (543 bp), two distinct forms of mouse TGF-beta type II receptor (526 bp for mTGF-beta receptor II1 and 601 bp for mTGF-beta receptor II2), and the IFN-{gamma} receptor 1 (660 bp) were expressed on single longitudinal muscle cells isolated from control mice (Fig. 1).


Figure 1
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  		Fig. 1. A: TGF-beta1 mRNA expression in longitudinal muscle layer from control (C) mice. One of three separate experiments that gave similar results is shown. M, size marker. B: mouse IFN-{gamma} receptor 1 (IFN-{gamma}R) and two distinct forms of mouse TGF-beta type II receptor (TGF-betaR) expression in dispersed longitudinal smooth muscle cells from control mice. One of five separate experiments that gave similar results is shown.

 
Ligand-binding characteristics of the muscarinic receptor on longitudinal smooth muscle cells. As shown in Fig. 2A, the specific binding of [3H]NMS to isolated longitudinal smooth muscle cells was concentration dependent over a range of 0.1–10 nM, and above this range binding was saturated. Scatchard analysis revealed a KD for [3H]NMS of 2.6 ± 0.7 nM and a Bmax of 2.4 ± 0.6 x 104 sites/cell, respectively, in control cells. Carbachol was used to identify agonist-binding characteristics. As shown in Fig. 2B, the competition binding curve identified a high-affinity (KH) and a low-affinity (KL) binding site, with Ki of 4.1 nM and 0.2 mM, respectively.


Figure 2
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  		Fig. 2. A: concentration dependence of [N-methyl-3H]scopolamine ([3H]NMS) binding. Cells were incubated with various concentrations of [3H]NMS for 15 min at room temperature. Specific binding is difference between [3H]NMS binding in presence or absence of atropine. Values are means of 4 experiments, each done in duplicate. B: inhibition of [3H]NMS binding by muscarinic agonist carbachol. Cells were incubated with 1 nM [3H]NMS for 15 min at room temperature in presence or absence of carbachol. Values are means of 5 experiments, each done in duplicate.

 
The effect of T. spiralis infection on the muscarinic receptor [3H]NMS binding to longitudinal smooth muscle cells. Infection was accompanied by a significant reduction in KD from 2.6 nM to 0.7 nM and a substantial decrease in KH from 4.1 nM to 0.05 nM (P < 0.05). There was also a reduction in KL from 0.2 mM to 0.02 mM. In contrast, there was no significant change in the Bmax between cells from control and infected mice (Table 1). Previously (1, 19), we had shown that the hypercontractility of muscle to carbachol in T. spiralis-infected mice is STAT6 dependent. Therefore, we next examined whether the presence of STAT6 alters the muscarinic receptor-binding characteristics. No differences were seen in either binding capacity of affinity constants on muscle cells from STAT6+/+ and STAT6–/– mice (Table 1).


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  		Table 1. [3H]NMS binding to intestinal longitudinal smooth muscle cells from control and STAT6–/– mice with or without Trichinella spiralis infection

 
Influence of cytokines on muscarinic receptor binding. Preincubation of cells with IL-4, IL-13, or IFN-{gamma} to LMMP had no significant effect on KD or Bmax for atropine. However, preincubation of cells with TGF-beta1 increased Bmax (5.0 x 104 sites/cell; P < 0.01). Two agonist-binding sites were identified in displacement experiments using carbachol: KL of 0.2 mM and KH of 4.1 nM in control mice. Preincubation of cells with IL-13 and TGF-beta1 decreased the KH to 0.2 nM and 2.2 pM, respectively. In contrast, preincubation with IFN-{gamma} increased the KH to 0.4 µM (Table 2). IL-13 and TGF-beta1 increased muscarinic receptor affinity, whereas Th1 cytokine IFN-{gamma} decreased the muscarinic receptor affinity. These cytokines did not change KL significantly.


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  		Table 2. [3H]NMS binding to intestinal longitudinal smooth muscle cells incubated with IL-4, IL-13, TGF-beta, and IFN-{gamma}

 
Effects of TGF-beta1 and IFN-{gamma} on longitudinal muscle contractility. To determine the effects of the TGF-beta1 and IFN-{gamma} on muscle contractility, LMMP were preincubated with 10 ng/ml of these cytokines overnight before dispersion of the cells and subsequent stimulation by carbachol. All carbachol-induced responses were abolished by 1 µM atropine (data not shown). Preincubation with TGF-beta1 and IFN-{gamma} did not alter mean cell length; cell length was 50.7 ± 1.0 and 55.3 ± 5.7 µm in TGF-beta1- and IFN-{gamma}-exposed cells, respectively, compared with 49.8 ± 6.3 µm in control cells. Carbachol (0.1 nM)-induced contraction of cells exposed to TGF-beta1 was 17.3 ± 1.0%, reflecting a 188% increase in contractility compared with control cells (P < 0.01). In addition, the half-maximal effective dose (ED50) for carbachol-induced contraction was 375-fold less in TGF-beta1-exposed cells compared with control (0.8 pM vs. 0.3 nM, respectively). On the contrary, 10 nM and 1 nM carbachol-induced contractions of cells exposed to IFN-{gamma} was 16.2 ± 2.4% and 9.1 ± 1.9%, reflecting a 31% (P < 0.05) and 54% (P < 0.01) decrease in contractility compared with control cells. In addition, the ED50 for carbachol-induced contraction was not significantly changed in IFN-{gamma}-exposed cells compared with control (0.5 nM vs. 0.3 nM; Fig. 3).


Figure 3
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  		Fig. 3. Dose-response curve of the carbachol-induced jejunal longitudinal smooth muscle cell contraction from noninfected control cells ({circ}), cells incubated with 10 ng/ml TGF-beta1 ({blacksquare}), and cells incubated with 10 ng/ml IFN-{gamma} ({square}). Values are means ± SE of 4–6 experiments. +P < 0.01 for cells incubated with IFN-{gamma} compared with control cells at same concentration. *P < 0.05 for cells incubated with TGF-beta1 compared with control cells at same concentration.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Inflammation of the gastrointestinal tract is accompanied by changes in smooth muscle contractility. A previous study of tissue from Crohn's disease patients (33) revealed hypercontractility of intestinal muscle mediated by muscarinic receptor activation. In a model of nematode-induced infection in mice, T cells infiltrating the muscularis externa produce the Th2 cytokines IL-4 and IL-13. IL-4 enhanced muscle contractility mediated by muscarinic receptors, and this was completely STAT6 dependent, whereas that induced by IL-13 was largely STAT6 independent in isolated smooth muscle cells that ligands affect directly (1). In contrast, in acetic acid-induced ileal inflammation suppressed the phasic contractile response to muscarinic receptor activation in canine circular muscle (26). In a murine model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid, which exhibits a predominant Th1 cytokine profile (24), acetylcholine-induced maximum active tension of circular muscle strips was transiently decreased in rabbit colon (9). Martinolle et al. (20) reported that whereas inflammation increased longitudinal muscle contraction, it decreased circular muscle contraction in response to histamine and carbachol in guinea pig model of trinitrobenzene sulfonic acid-induced ileitis. In contrast, AKR mice infected with Trichuris muris generate a Th1 immune response, resulting in hypocontractility of gut longitudinal muscle and markedly delayed expulsion of the parasites (22). The inflammation-induced intestinal motility may be species or tissue specific. Together, these observations suggest that Th1 and Th2 cytokines may have opposing effects on murine longitudinal smooth muscle contraction mediated by muscarinic agonists.

In this study, we used longitudinal muscle cells to demonstrate the expression of the cytokine receptors TGF-beta receptor and IFN-{gamma} receptor, which mediate the effects of TGF-beta1 and IFN-{gamma}, respectively. We have shown (1) the mRNA expression for IL-4 receptor {alpha} on the dispersed longitudinal muscle cells, which IL-4 and IL-13 can bind. TGF-beta exerts its multiple actions through two types of transmembrane receptors (type I and type II) (21). IFN-{gamma} exerts its effects through the IFN-{gamma} receptor, which is composed of two distinct subunits, IFN-{gamma} receptor 1 and IFN-{gamma} receptor 2. The major ligand-binding subunit is IFN-{gamma} receptor 1, which plays an important role in IFN-{gamma} signaling (4, 27).

Whereas the binding study showed both high- and low-affinity binding sites with Ki values of 4.1 nM and 0.2 mM, respectively, the ability of muscle cells to shorten on exposure to nanomolar concentrations of the muscarinic agonist carbachol suggests that the high-affinity binding site is linked to contraction. In addition, it was only the high-affinity binding site that was altered during T. spiralis infection or cytokine exposure, conditions known to alter the contractility of the tissue.

Results in the receptor-binding study showed that infection was accompanied by a reduction in KD in both STAT6+/+ and STAT6–/– mice, suggesting that infection increased muscarinic receptor affinity via a STAT6-independent pathway to evict the parasites. In competition experiments, there was a reduction in KH in both STAT6+/+ and STAT6–/– mice during infection. No significant differences in KL and KH were seen between uninfected STAT6+/+ and STAT6–/– mice. These results demonstrate that T. spiralis infection altered the muscarinic receptor affinity for carbachol in both STAT6–/– and STAT6+/+ mice and suggest that infection increased only the high-affinity site of the muscarinic receptor via a STAT6-independent pathway. During T. spiralis infection in mice, the expression of IL-4 and IL-13 mRNA in the intestinal longitudinal muscle layer was significantly increased compared with controls. In contrast, there was no significant change in IFN-{gamma} mRNA expression in the longitudinal muscle layer (19). TGF-beta1 mRNA expression in the muscle increased during infection (data not shown). In a recent study (1), we showed that exposure of murine longitudinal muscle from STAT6–/– mice to IL-13 but not IL-4 enhanced carbachol-induced longitudinal muscle cell contraction. In the present study, exposure of longitudinal muscle to TGF-beta1, but not IFN-{gamma}, enhanced contractility. Together, these results are consistent with the hypothesis that the Th2 cytokine IL-13 and the regulatory cytokine TGF-beta1 act via their receptors on longitudinal smooth muscle to increase the affinity of the muscarinic receptor and activate postreceptor signaling pathways, resulting in a hypercontractile state. This is supported by direct binding studies showing that preincubation of cells with IL-13 and TGF-beta1 decreased the value of KH, whereas preincubation with IFN-{gamma} increased the value of KH.

Interactions between Th1 or Th2 cytokine and muscarinic receptors in other systems have also been reported. Fryer et al. (13) reported that in asthma, there is decreased M2 muscarinic receptor expression on cholinergic nerves. These neuronal M2 receptors inhibit acetylcholine release. Thus the reduced expression of these receptors induces bronchoconstriction via increased release of acetylcholine acting on M3 receptors on muscle. Jacoby et al. (17) showed the similar results. In this study, only TGF-beta1 increased the number of muscarinic receptors, and TGF-beta1 increased muscarinic receptor affinity more than IL-13. TGF-beta1 has also been shown to downregulate M2 muscarinic receptor protein and mRNA in human embryonic lung fibroblasts (14) as well as to downregulate of M2 and M4 muscarinic receptor in chick heart cells (16). Thus the effects of cytokines on muscarinic receptors are tissue specific.

Studies in asthma have identified Th2 cells and their cytokines in the development of airway hyperreactivity in atopic and intrinsic asthmatics (3, 35, 15). In a canine model of asthma, glucocorticoid treatment decreased M2 and M3 muscarinic receptor expression in airway smooth muscle (12), again suggesting that there is an association between immune responses and muscarinic receptor activation during airway inflammation. In conclusion, this study clearly indicates that cytokines exert differential effects on the muscarinic receptor on intestinal longitudinal smooth muscle and explain the basis for altered muscle contractility observed in Th1 and Th2 models of inflammation, as well as in the post-nematode-infected state.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Canadian Institutes of Health Research (to S. M. Collins) and by a Research Initiative Award from the Canadian Association of Gastroenterology and AstraZeneca (to H. Akiho).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Akiho, Dept. of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu Univ., 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (e-mail: akiho{at}intmed3.med.kyushu-u.ac.jp)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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