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* Department of Medicine and Mucosal Biology Research Center, University of Maryland School of Medicine, Baltimore, MD 21201;
Nutritional Requirements and Function Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705;
Laboratory of Veterinary Pathology, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan;
Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
¶ Department of Medicine and Department of Pediatrics, University of Cincinnati, Cincinnati, OH 45267;
|| Cincinnati Veterans Administration Medical Center, Cincinnati, OH 45220; and
# Division of Parasitology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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1 component of the type 2 IL-4R that is linked to STAT6. IL-13 also binds to the IL-13R2 that is not linked to STAT6 and functions to limit the effects of IL-13 in vivo. In this study we assessed the contributions of regional and cellular differences in the distribution of the IL-13R components to the physiological regulation of smooth muscle function in wild-type mice and mice deficient in STAT6 or IL-13R2. The expression of IL-13 and IL-13R2 was higher in colon than in small intestine. Laser capture microdissection of specific cell types revealed that the expression of IL-13R2 was higher in the smooth muscle layer compared with levels in the epithelial cells of the mucosa. In contrast, there was a uniform distribution of IL-131 in smooth muscle, epithelia, and myenteric neurons. The significant hypercontractility of smooth muscle in mice deficient in IL-13R2, but not in STAT6, shows the physiological importance of IL-13 binding to IL-13R2. The pronounced differences in the expression of IL-13R2 suggest that the gut has developed sophisticated mechanisms for controlling the physiological and pathophysiological activities of IL-13. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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IL-4 and IL-13 share immunologic and biologic functions that can be explained in part by their sharing a receptor component. It is reported that there are at least three different receptors that bind IL-4 and/or IL-13. The type 1 receptor is composed of the common IL-2R 
-chain and the IL-4R 1-chain, whereas the type 2 receptor is composed of the IL-4R 1- and IL-13R 1-chains. Binding of IL-4 to the type 1 or type 2 receptor or of IL-13 to the type 2 receptor leads to phosphorylation of the IL-4R -chain and activation of the transcription factor STAT6. Th2 cytokines must bind to their receptors to induce biological responses; therefore, the location and level of expression of these receptors are of interest.
A second IL-13R chain, IL-13R2, reportedly binds IL-13 with high affinity, but is not linked with STAT6 signaling (5). The function of IL-13R2 remains unclear, but previous studies demonstrated that soluble IL-13R2 in serum acts as a decoy receptor to limit the actions of IL-13 in vivo (6, 7). Despite the number of studies demonstrating the potent effects of Th2 cytokines on the gastrointestinal tract, there is little information on the locations of these receptors, particularly on nonhemopoietic effector cells, such as epithelial cells, smooth muscle cells, and enteric neurons. The aim of the present study was to determine in the intestine and colon 1) the regional differences in the constitutive expression of the cytokine receptors for IL-4 and IL-13, 2) the expression of cytokine receptors in specific cell types harvested by laser capture microdissection, and 3) the functional importance of IL-13R2. The results of the present study are the first to demonstrate regional as well as cell-specific differences in cytokine receptor distribution that potentially impact Th2 effects on gut function. In addition, they support a phenotype for IL-13R2 in the physiological regulation of endogenous IL-13 on gut motility.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Tissues were taken from BALB/c mice, 8–12 wk old (National Cancer Institute), mice deficient in the expression of STAT6 (STAT6–/–)), IL-4 (IL-4–/–; both from F. Finkelman), IL-13R2 (IL-13R2–/– from T. Wynn, or IL-13–/– from W. Paul), to assess the contribution of IL-13 acting though STAT6-dependent and -independent receptors. Serum levels of IL-13R2 were determined as described previously (8).
Preparation of tissue, frozen blocks, and sectioning
Segments of tissue were taken from the mid small intestine (jejunum) and colon, and one piece was homogenized in TRIzol (Invitrogen Life Technologies) and stored at –80°C until needed. A second section was slit longitudinally, laid flat with the mucosal surface up, rolled around a wood stick (Swiss roll), and embedded in Tissue-Tek OCT compound (Sakura Finetek) in cryomold. The tissues were frozen on dry ice-acetone, removed from the cryomold while still frozen, and placed at –80°C in an airtight container until sectioned (HM505E cryostat; Richard-Allan Scientific). For laser capture microdissection (LCM)4, 4-µm tissue sections were made from frozen blocks and placed immediately on plain uncoated slides on dry ice, then stored at –80°C until needed. Sections of frozen tissue were stained with H&E to assess changes in tissue morphology (ECLIPSE TE2000E; Nikon Instruments). For each mouse, the thickness of the muscle layer was assessed in four different architecturally normal sites in each region and then averaged to obtain one value (micrometers) per region.
Laser capture microdissection
LCM was performed as described previously (9, 10). Briefly, cryosectioned tissue was stained with H&E and dehydrated. LCM was performed on a PicCell II (Arcturus Engineering), and target cells were transferred to CapSure LCM Caps (Arcturus Engineering). Cells were captured from the region of the epithelium, lamina propria, muscles, and myenteric plexus in small intestine and colon.
Gene expression analysis (RNA extraction, RT, and real-time PCR)
Total RNA extraction from whole tissue was performed according to the manufacturer’s instructions. RNA was extracted from LCM samples, and RT was performed as described previously (9, 10). Primers and probes for IL-13, IL-4, STAT6, IL-4R1, IL-13R1, and IL-13R2 were designed using Primer Express software (Applied Biosystems) with sequences obtained from GenBank as follows: IL-4, 5'-CGGAGATGGATGTGCCAAAC-3' (sense), 5'-GCACCTTGGAAGCCCTACAG-3' (antisense), and 5'-TCCTCACAGCAACGAAGAACACCACA-3' (probe); IL-13, 5'-GACCAGACTCCCCTGTGCAA-3' (sense), 5'-TGGGTCCTGTAGATGGCATTG-3' (antisense), and 5'-CGGGTTCTGTGTAGCCCTGGATTCC-3' (probe); IL-4R1, 5'-CCCACCTGCTTCTCTGACTACAT-3' (sense), 5'-GTTTTCAGAGAACTCGAAGAACATCA-3' (antisense), and 5'-ACTGCAGTTCTCAGCTCTGCCTACACTACAGG-3' (probe); IL-13R1, 5'-CATGGAGGGTACAAGTTG-TTTCC-3' (sense), 5'-GTTTTGACTCTTACTCTGACTGTGTAGACA-3' (antisense), and 5'-CTCCCTGGTGTTCTTGCCGACGC-3' (probe); IL-13R2, 5'-TGTCTTTTCTTTATATTCCTTTTGTTACTTCT-3' (sense), 5'-ACACACTTCTTTGTTCAGATCCACAT-3' (antisense), and 5'-AAGAACCTGAACCCACATTGAGCCTCCA-3' (probe); and STAT6, 5'-GACTATGGTAGAGGACAGTTGCCTAA-3' (sense) and 5'-GGTTCTCCAGGAGAAGCTTGGT-3' (antisense).
Real-time PCR was performed for STAT6 using the SYBR Green Supermix (Bio-Rad) with iCycler (Bio-Rad). For all other probes, real-time PCR was performed using the Brilliant Quantitative PCR Core Reagent kit (Stratagene) on an ABI PRISM 7700 Sequence Detector System (Applied Biosystems). Amplification conditions were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Fluorescence signals measured during amplification were processed after amplification. Data for whole tissue were normalized to 18S rRNA and then expressed as the fold change in mRNA expression compared with wild type (WT) (Applied Biosystems). For LCM samples, each experiment was repeated three times (n 
3 each time).
Smooth muscle contractility
Segments of jejunum or mid-colon (1 cm) were flushed of their intestinal contents, suspended longitudinally in individual 8-ml organ baths, and maintained in oxygenated Krebs solution at 37°C. One end of the tissue was attached to an isometric tension transducer (model FT03; Grass Medical Instruments), and the other to the bottom of the bath. Tissues were stretched to a load of 9.9 mN (2 x g), because preliminary experiments showed that this load stretched tissues to their optimal length for active contraction (11). Tissues were allowed to equilibrate for at least 30 min in Krebs buffer solution before concentration-response curves to acetylcholine, a major neurotransmitter in the gut (1 nM to 100 µM), were constructed. The bath solution was replaced every 10 min throughout each study. Tension was recorded using a Grass model 79 polygraph and was expressed as force per cross-sectional area (12).
Statistics
Concentration-dependent responses to acetylcholine were compared using MANOVA (Systat 5.2) with post-hoc analysis for multiple comparisons. A value of p < 0.05 was considered significant. Differences in mRNA expression among the different treatment groups were determined using one-way ANOVA, followed by Tukey’s test. Appropriate vehicle and time- and age-matched controls were performed for each group (n = 3–5/group). There were no significant differences among WT controls; therefore, data were pooled.
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Results |
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To investigate differences in the expression of IL-4 and IL-13 between small intestine and colon, sections of whole tissue were taken from WT mice, and RNA was extracted and analyzed using real-time PCR. IL-4 expression in the small intestine and colon was low; however, IL-13 expression was significantly higher in the colon than in the small intestine (Fig. 1A). In addition, STAT6 expression was 
3-fold higher in the small intestine than in the colon (small intestine, 1.00 ± 0.12; colon, 0.33 ± 0.03). A comparison of cytokine receptor expression in whole tissue showed little difference in the expression of IL-4R or IL-13R1 between the small intestine and colon, but a striking difference in IL-132 expression between the two areas, with a much higher expression in the colon (Fig. 1B). This high expression of IL-13R2 in colon was unexpected and was investigated further.
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2 gene expression
To determine the physiological regulation of IL-13R2 gene expression, full-thickness sections of intestine and colon were taken from WT, IL-4–/–, and IL-13–/– mice. Because the expression of IL-13R2 was constitutively low in the intestine (Fig. 1B), we did not perform additional analyses in deficient mice. In contrast, the high constitutive expression of the decoy receptor in the colon was inhibited significantly in IL-4–/– mice and to an even greater extent in IL-13–/– mice (Fig. 2).
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There are limits to the interpretation of observations in whole tissue, and given the heterogeneity of cell types along the gut, expression is not likely to be uniform in all cell types on specific nonimmune cells. To identify microenvironments where the receptors are expressed, we used LCM to rapidly isolate specific cell populations or regions from tissue sections on a microscope slide. Cells were captured from epithelium, lamina propria, smooth muscle, and myenteric plexus in small intestine and colon. Total RNA was isolated from LCM samples, converted to cDNA, and IL-4R, IL-13R1, IL-13R2, and ribosomal RNA were assessed by real-time PCR. All values were normalized to constitutive ribosomal RNA values.
We showed previously that, with the exception of Peyer’s patches, IL-13 and IL-4 gene expression in a number of cell types was very low in uninfected mice (10). Constitutive expression of IL-4R in normal mice was low in epithelial cells of the intestine and colon (Table I). Smooth muscle expression was higher than that in the epithelia, but was similar in both regions, whereas expression in myenteric plexus was much greater in the colon relative to the small intestine. IL-13R1 expression levels in the epithelium were greater in the colon than in the small intestine, but smooth muscle and myenteric plexus were similar in both regions (Table I). Finally, the expression of IL-13R2 in small intestine was very low, with detectable levels only in smooth muscle, correlating with the low levels observed in whole tissue (Fig. 1B). In contrast, IL-13R2 expression was dramatically higher in colonic smooth muscle (Fig. 3). These levels in the colon were cell specific, because there was no IL-13R2 expressed in the epithelial cells or myenteric plexus, demonstrating the value of LCM for probing selective cell types.
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Enteric helminth infection induced a STAT6-dependent hypercontractility of smooth muscle, demonstrating a role for Th2 cytokines (11). In normal mice, however, there were no differences in response to acetylcholine between WT and STAT6–/– mice in either the intestine (Fig. 4A) or colon (Fig. 4B) despite regional differences in the expression of STAT6. IL-13 has a greater effect on smooth muscle than IL-4 (11), suggesting a greater contribution of IL-13 to helminth-induced hypercontractility. To investigate the contribution of the IL-13R2, which is not linked to STAT6, to the physiological control of gut function, smooth muscle responses to acetylcholine were evaluated in IL-13R2–/– mice. In both the small intestine and colon, responses to acetylcholine were elevated significantly in IL-13R2–/– mice compared with WT (Fig. 4) in the absence of any change in smooth muscle thickness (small intestine, 41.0 ± 6.2 vs 34.8 ± 2.2 µm; colon, 107.6 ± 14.1 vs 134.8 ± 10.2 µm). This is the first demonstration of a change in constitutive gut function in the absence of a specific cytokine receptor, IL-13R2–/–.
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2–/– mice
Serum concentrations of IL-13R2 were undetectable in IL-13R2–/– mice compared with WT mice (7.2 ± 4.9 ng/ml). To determine whether the enhanced response to acetylcholine in IL-13R2–/– mice could be attributed to changes in cytokine gene or other cytokine receptor components, the expressions of IL-4, IL-13 gene, as well as IL-4R and IL-13R1 in intestine and colon were determined by real-time PCR. There were no differences in the expression of either IL-4 or IL-13 in the intestine of IL-13R2–/– mice (Table II). In contrast, in the colon, there was a 3-fold greater expression of IL-4 and a 20-fold greater expression of IL-13 in IL-13R2–/– mice compared with WT mice. The expression of the receptor components, IL-4R and IL-13R1, was similar in WT and IL-13R2–/– mice (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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2. In the absence of IL-13R2, there is a smooth muscle hypercontractility and an elevation in IL-13 gene expression in both regions in the absence of significant alterations in smooth muscle morphology. In addition, IL-13R2 expression in uninfected mice is regulated in part by IL-13 and IL-4. These data are the first to demonstrate roles for endogenous IL-13 and IL-13R2 in the physiological regulation of smooth muscle function in the gut.
IL-4 and IL-13 share a receptor component as well as some immunological functions, but they also exhibit different biological effects. Helminth-induced alterations in epithelial cell function and smooth muscle contractility in the small intestine are STAT6-dependent (11, 14, 15) and are mimicked by exogenous IL-4 and IL-13, indicating that these effects are due in part to activity at the type 2 receptor. Studies of the exogenous administration of IL-4 or IL-13 reveal that these cytokines have remarkably similar effects on sodium-linked glucose absorption and tissue permeability, but that there are also several differences, such as the prosecretory effects of IL-4 in the small intestine, which are mast cell-dependent and are not shared by IL-13 (16). In addition, IL-13 has a greater effect than IL-4 on intestinal smooth muscle contractility (11). Some of these differences between IL-4 and IL-13 on gut function can be explained by differences in the structure and expression of their receptors. Although both receptors are linked to STAT6 signaling through the IL-4R -chain, the type 1 receptor is expressed predominantly by bone marrow-derived cells and binds IL-4, but not IL-13. The tissue distribution of type 1 and type 2 receptors, therefore, will influence responses to IL-4 vs IL-13.
In the present study there were striking regional variations in cytokine and cytokine receptor gene expression. STAT6 expression was higher in the small intestine than in the colon, yet there were no differences between WT and STAT6–/– mice in the contractile response to acetylcholine. This is consistent with previous reports showing the lack of a constitutive effect of STAT6 signaling on smooth muscle (11) or epithelial cell responses (16) and suggests that other factors influence the activities of IL-13 and IL-4. IL-13 gene expression was markedly higher in the colon compared with the small intestine. Although the expression of IL-4R and IL-13R1 was similar in both areas, there was a predominant expression of IL-13R2 RNA in the colon. The contribution of IL-13R2 to cytokine effects in the gut is unknown, but evidence suggests that soluble IL-13R2 in the serum functions as a decoy receptor to limit the effects of IL-13 in vivo. In this paradigm, responses to IL-13 should be enhanced in the absence of IL-13R2. There are convincing data showing an elevated responsiveness to IL-13 in IL-13R2–/– mice marked by elevated production of IgE and increased macrophage progenitors (7) as well as an exaggerated liver fibrosis in Schistosoma mansoni-infected mice (6). This was attributed to an apparent movement of IL-13 from serum to tissue (6). This is consistent with the results of the present study showing that IL-13R2–/– mice have undetectable levels of the receptor in serum and exhibited a significant hypercontractility of smooth muscle in both the intestine and colon. This could not be attributed to changes in the tissue expression of other receptor components, IL-4R or IL-13R1. It is important to note that the effects of IL-13 on smooth muscle contractility as well as on epithelial cell function are dependent largely on STAT6. In the intestine and colon, therefore, the functional effects of IL-13 are mediated by binding to IL-13R1, which is part of the type 2 receptor containing IL-4R that is linked to STAT6, whereas the constitutive levels of IL-13R2 act to limit the amount of IL-13 available to bind to IL-13R1. The greater movement of IL-13 to the tissue in IL-13R2–/– mice elicited a comparable hypercontractility in the small intestine and colon because of the similar expression of IL-13R1.
If the level of IL-13R1 limits the functional impact of IL-13 in the gut, what is the role for IL-13R2? The gut exhibited pronounced regional differences in IL-13R2 RNA expression, with a markedly higher expression in the colon. LCM analysis of specific cell types revealed that RNA expression of IL-13R2 was low in mucosal epithelial cells in both regions, but was constitutively expressed in smooth muscle cells, particularly in the colon. This regional and cellular distribution of IL-13R2 suggests that there may be a greater need for the decoy receptor in the colon, because constitutive IL-13 gene expression is higher in this area. This is supported by the observation that in the absence of IL-13R2–/–, the expression of IL-13 was elevated markedly above that in WT mice, but only in the colon. The immune regulation of this receptor is demonstrated further by the finding that the constitutive expression of IL-13R2 in the colon is regulated by IL-13 and, to a lesser extent, by IL-4. This cytokine- and region-specific effects on gene expression imply that in the colon, IL-13 positively regulates IL-13R2 expression, which, in turn, negatively regulates IL-13 gene expression, and that there is a physiological reason for limiting effects of IL-13 in this region. In response to certain enteric helminth infections, IL-13 can functionally replace IL-4 and initiate parasite expulsion (18). IL-13 has potent effects on mucosal permeability and smooth muscle contractility in the small intestine that are part of the host response to increase intraluminal fluid accumulation and promote motility to facilitate helminth expulsion. Comparable permeability changes in the colon would be detrimental given the bacterial load in this region and the need to maintain the high mucosal resistance required in this area for ion and fluid absorption. Although the source of soluble IL-13R2 is unclear, these data also suggest that the colon may serve as a constitutive source of this receptor that can be amplified during helminth infection. The restriction of IL-13R2 expression to smooth muscle may serve to limit the effects of IL-13 on the adjacent mucosa.
In summary, these data are the first to show the functional importance of the regional- and cell-specific differences in the distribution of cytokine gene and receptor expression in the gastrointestinal tract. Despite the importance of IL-13 in protective immunity, however, the prominent differences in the expression of IL-13R2, but not IL-13R1, suggest that the gut has developed sophisticated mechanisms for controlling or focusing the physiological and pathophysiological activities of IL-13. Disruption of this balance may have both physiological and clinical relevance.
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported in part by National Institutes of Health Grant RO1AI/DK49316 (to T.S.-D.) and U.S. Department of Agriculture CRIS 1235-52000-053 (to J.F.U.). The opinions and assertions in this article are those of the authors and do not necessarily represent those of the U.S. Department of Defense or the U.S. Department of Agriculture. 
2 Mo.M. and Ma.M. contributed equally to this project.
3 Address correspondence and reprint requests to Dr. Terez Shea-Donohue, Mucosal Biology Research Center, University of Maryland School of Medicine, 20 Penn Street, HSFII, Room S351, Baltimore, MD 21201. E-mail address: tdonohue{at}mbrc.umaryland.edu
4 Abbreviations used in this paper: LCM, laser capture microdissection; WT, wild type.
Received for publication April 12, 2005. Accepted for publication October 3, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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2: molecular cloning, characterization, and comparison with murine IL-13 receptor 1. J. Immunol. 161: 2317-2324. 2 during a T helper cell type 2-dominant immune response. J. Exp. Med. 197: 687-701. 2. J. Exp. Med. 197: 703-709. 2 down-modulates granulomatous inflammation and prolongs host survival in schistosomiasis. Proc. Natl. Acad. Sci. USA 101: 586-590. , and Stat6 are required for the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity 8: 255-264. [Medline]This article has been cited by other articles:
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  J. Wang, L. Chen, P. Li, X. Li, H. Zhou, F. Wang, D. Li, Y. Yin, and G. Wu Gene Expression Is Altered in Piglet Small Intestine by Weaning and Dietary Glutamine Supplementation J. Nutr., June 1, 2008; 138(6): 1025 - 1032. [Abstract] [Full Text] [PDF]
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 Y. Tabata, W. Chen, M. R. Warrier, A. M. Gibson, M. O. Daines, and G. K. K. Hershey Allergy-Driven Alternative Splicing of IL-13 Receptor {alpha}2 Yields Distinct Membrane and Soluble Forms J. Immunol., December 1, 2006; 177(11): 7905 - 7912. [Abstract] [Full Text] [PDF]
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