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Am J Physiol Gastrointest Liver Physiol 292: G385-G394, 2007; doi:10.1152/ajpgi.00065.2006
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NEUROREGULATION AND MOTILITY

IL-4 gene transfer to the small bowel serosa leads to intestinal inflammation and smooth muscle hyperresponsiveness

Bruce A. Vallance,1 Nicola Radojevic,2 Cory M. Hogaboam,5 Yikang Deng,2 Jack Gauldie,3 and Stephen M. Collins2,4

1Division of Gastroenterology, British Columbia’s Children’s Hospital, Vancouver, British Columbia; 2Intestinal Diseases Research Program and Departments of 3Pathology and 4Medicine, McMaster University, Hamilton, Ontario, Canada; and 5Department of Pathology, University of Michigan, Ann Arbor, Michigan

Submitted 8 February 2006 ; accepted in final form 11 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intestinal mucosal inflammation can lead to altered function of the underlying smooth muscle, which becomes hyperreactive to most contractile stimuli. Through nematode parasite infection models, T helper type 2 (Th2) cytokines have been implicated in intestinal muscle dysfunction; however, the mechanisms involved and the relevance of these findings to other forms of intestinal inflammation are unclear. Through gene transfer, we explored whether the Th2 cytokine IL-4 can mediate changes in longitudinal muscle function in the context of an adenoviral infection. Following abdominal surgery on mice, control beta-galactosidase-encoding recombinant adenoviruses and IL-4-encoding adenoviruses were applied to the serosal surface of the jejunum, leading to infection of cells in the serosa and in the mesentery. Marker transgene expression lasted for 3 wk and was accompanied by the recruitment of macrophages, lymphocytes, and neutrophils into the peritoneal cavity and mild inflammation at the site of infection. IL-4 transgene expression led to a stronger inflammatory response characterized by tissue eosinophilia and increased numbers of peritoneal mast cells and plasma cells. Whereas control virus infection had no effect on intestinal muscle function, infection with the IL-4 virus led to significant jejunal muscle hypercontractility, evident by day 7 postinfection. This modulation of smooth muscle function was shown to be IL-4 specific, since the application of an IL-5-encoding adenovirus induced tissue eosinophilia but did not alter muscle function. These results highlight an important causal role for IL-4 in the pathological regulation of enteric smooth muscle function and identify a novel strategy for gene transfer to the intestine.

adenovirus; immunomodulation; motility; enteric infections; pathophysiology; interleukin-4

PREVIOUS STUDIES have shown that inflammatory events in the intestinal mucosa lead to altered function of the underlying smooth muscle, which becomes hyperreactive to most contractile stimuli (9, 38, 44). Changes in intestinal muscle contractility have been demonstrated in inflammatory bowel diseases (IBD) (35, 46) and during many forms of enteric infection (9, 10, 33) and are thought to contribute to the pain, nausea, and diarrhea associated with gastrointestinal (GI) inflammation. Because of the complex nature of these inflammatory conditions, the underlying mechanisms remain unclear. To identify the mediators involved in the pathogenesis of intestinal muscle dysfunction, studies have focused on models of Trichinella spiralis and Heligosomoides polygyrus infection in rodents (13, 14, 22, 38, 44). These nematode parasites trigger intestinal inflammation, and, through the manipulation of the resulting host immune response, we and others have identified a strong association between T helper type 2 (Th2) cytokines and intestinal smooth muscle hyperresponsiveness. Based on these studies, a central role for the cytokine IL-4 has been hypothesized in the regulation of enteric smooth muscle function during inflammation (14, 52). In support of this hypothesis, both IL-4-deficient mice and STAT6-deficient mice are impaired in the characteristic generation of muscle hypercontractility following nematode infection (22, 52), whereas pretreatment of smooth muscle cells with IL-4 increases their contractile response (2, 4). Unfortunately, it is unclear whether the increased expression of IL-4 is sufficient on its own to alter muscle function in vivo or if IL-4 possesses such actions in the context of other forms of infectious or idiopathic intestinal inflammation.

With these goals in mind, we explored the potential of using gene transfer technology to overexpress IL-4 in the intestine. Gene transfer and the ability to direct the site of transgene expression have potential for studying both the physiological and pathophysiological roles of these genes within specific tissues (34, 42) as well as possible medical applications in the treatment of diseases through the ectopic expression of therapeutic proteins (48). Recombinant adenoviruses are the most efficient and extensively used vectors currently available for gene transfer in vivo. Infectious across a broad range of host species and cell types, adenovirus vectors can be grown to high titers, and, following infection, they direct the cellular machinery of host cells to produce large quantities of transgene proteins (16). Beyond their potential therapeutic application in the correction of genetic defects and autosomal recessive diseases such as cystic fibrosis (48), recombinant adenoviruses have also found use in the study of immune and inflammatory responses. Many studies have used recombinant adenoviruses incorporating cytokine or growth factor genes to study the effects of transient expression of these immunomodulatory molecules on inflammation (7, 51), fibrosis (25, 34), and host defense (17).

Unfortunately, while many advances have been made in the field of gene transfer to the lung and other tissue sites, intestinal gene transfer is still in its infancy (18, 32). In vivo infection of intestinal epithelial cells, through lumenal or oral introduction of viral vectors, has been attempted by several groups; however, the innate protective barriers found in the GI tract drastically limit transduction efficiency (50). While we and others have found that transient disruption of colonic epithelial and mucus barriers can improve the transfection efficiency of adenovectors delivered by enema (42, 49), even allowing an adenovirus encoding transforming growth factor (TGF)-beta1 to cause colonic fibrosis (42), overexpression of the cytokines IL-4 and IL-12 through this route caused a fatal colitis (43). The high mortality resulting from this approach ruled out luminal-delivered adenoviral gene transfer as a means to study the role of IL-4 in muscle dysfunction; therefore, alternative approaches to intestinal gene transfer, particularly focused on impacting the outer muscle layers, needed to be developed and tested.

The aim of this study was therefore to locally overexpress IL-4 in close proximity to the enteric neuromuscular apparatus of mice. The muscularis externa was targeted through the application of adenoviral vectors onto the serosal surface of the mouse small intestine using a pluronic gel. Biocompatible pluronic gels have been previously shown to improve the efficiency of adenoviral gene transfer to endothelial and vascular smooth muscle cells both in vitro (26) and in vivo (12). In the present study, this approach led to the infection of large numbers of serosal cells and cells in the intestinal mesentery, as assessed by histochemistry for the marker virus-encoded beta-galactosidase (LACZ) enzyme. While this control virus triggered a modest inflammatory response involving neutrophils, macrophages, and lymphocytes, an IL-4-encoding virus led to a stronger inflammatory response with its cellular composition shifted to one typical of allergic inflammation, including numerous eosinophils, plasma cells, and mast cells. The inflammation in the IL-4 virus-infected mice was accompanied by a striking jejunal longitudinal muscle hyperresponsiveness that was not seen in AdLACZ-infected mice. This modulation of muscle function was specific to IL-4, since it was not seen following application of an IL-5-encoding virus despite the induction of significant tissue eosinophilia. These results thus highlight an important causal role for IL-4 in the pathological regulation of enteric smooth muscle function and identify a novel strategy for gene transfer to the intestine.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Specific pathogen-free, male C57BL/6 mice (6–10 wk old) were purchased from Taconic (Germantown, NY) and kept in filter-topped cages and given ad libitum access to autoclaved food and water before and during the study. The protocols employed were in direct accordance with the guidelines of the McMaster University Animal Care Committee and the Canadian Council on the Use of Laboratory Animals.

Recombinant adenovirus vectors. Recombinant human type 5 adenovirus incorporating mouse IL-4 cDNA into the E3 region of the viral genome (Ad5CAIL-4) has been previously constructed and well characterized in vitro and in vivo (1). This recombinant virus possesses the human cytomegalovirus (hCMV) promotor and polyadenylation signal sequences upstream and downstream of inserted IL-4 cDNA, respectively. Adenovirus incorporating Escherichia coli beta-galactosidase cDNA (Ad5LACZ) in the E3 region was constructed in a similar fashion (6) as was pACCMVmIL-5 adenovirus, which encoded murine IL-5 cDNA (47). High titers of viral constructs were generated as previously described (6). Virus was then purified by CsCl gradient centrifugation, dialyzed thoroughly as previously described (1, 6), aliquoted, and stored at –70°C until use.

Surgery study protocol and the establishment of viral infection. Mice were anesthetized by an intraperitoneal injection of ketamine and xylazine (70 and 6 ng/g body wt, respectively). Following a midline abdominal incision, the small bowel was gently exposed and covered with saline soaked gauze. A filter-sterilized gel solution containing 20% (wt/vol) F127 pluronic gel (BASF, Wyandotte, MI) in sterile PBS (pH 7.4) was mixed with 2 x 108 plaque-forming units (pfu) of adenovirus to give a final gel concentration of 15% (wt/vol) in a total volume of 80 µl. The virus-gel mixture was then gently dabbed onto a 6- to 8-cm region of the jejunum using a yellow pipette tip, beginning distal to the ligament of Trietz. The bowel was then covered with moist gauze and left for 5 min to allow the gel to set. Intestinal peristalsis was still present in all animals following the procedure. The small bowel was then gently returned to the abdominal cavity, and the incision was closed with discontinuous sutures. Following surgery, the animals were allowed to recover and then euthanized over the next 3 wk for tissue and peritoneal lavage collection.

Histochemical localization and enumeration of LACZ-expressing cells. Staining for LACZ expression was as described by Mastrangeli et al. (27) with minor modifications. Briefly, over the time course of the infection, mice were euthanized, and their small bowels were removed, flushed of lumenal contents, and then fixed with 2% formaldehyde in PBS at 4°C for 1 h. The intestine was then rinsed twice with PBS and immersed in staining solution containing 5 mM K4Fe(CN)6, 5 mM K3Fe3(CN)6, 2 mM MgCl2, and 0.5 mg/ml of X-gal stain (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, Boehringer Mannheim, Indianapolis, IN) at 37°C overnight. To enumerate the number of serosal cells expressing LACZ, the region of viral application was examined using a dissecting microscope. The numbers of blue-stained cells were determined per high-power field (x10 magnification) and averaged for 10 fields/tissue, with at least 4 mice examined per time point. For histological identification of the cells expressing LACZ, stained intestinal tissues were then paraffin embedded, sectioned at 6 µm, and counterstained with nuclear fast red.

Detection of IL-4 by RT-PCR. For analysis of IL-4 mRNA expression, LACZ- and IL-4 virus-infected mice were euthanized 3 days after infection, and the longitudinal muscle and myentric plexus (LMMP), including the serosa, was stripped from the infected jejunum. For in vitro analysis, murine smooth muscle cultures were prepared as previously described (19) and infected at a concentration of 10 pfu adenovirus/cell. Cells were harvested 3 days later. Total cellular RNA was isolated using the previously described guanidium isothiocyanate method (21). The RNA concentration was determined by measuring absorbance at 260 nm, and its purity was confirmed using the ratio of absorbency at 260 nm to that at 280 nm. mRNA was then used for RT-PCR as previously described (21) to yield cDNA. Aliquots (2 µl) of cDNA (3 µg) were then mixed with 20 pmol each of sense (5'-GAA TGT ACC AGG AGC CAT ATC-3') and antisense (5'-CTC AGT ACT ACG AGT AAT CCA-3') primer for murine IL-4 (36). The housekeeping gene was hypoxanthine phosphoribosyl transferase (HPRT), and 20 pmol of each sense (5'-GTT GGA TAC AGG CCA GAC TTT GTT G-3') and antisense primer (5'-GAT TCA ACT TGC GCT CAT CTT AGG C-3') were used to detect it (36). IL-4 and HPRT were coamplified for 33 cycles, and PCR products were visualized as previously described (20). The 383-bp product corresponded to IL-4, and the 164-bp product corresponded to HPRT.

Peritoneal lavage. Following euthanization, mice underwent peritoneal lavage, which involved the injection of 4 ml of cold PBS into the peritoneal cavity of each mouse. After 30 s of abdominal "massage," the abdominal cavity was opened, and the fluid was recovered and placed on ice. One milliliter of the fluid was centrifuged at 1,000 rpm for 8 min. The supernatant was then saved and stored at –70°C for the assay of IL-4. Another 1 ml was used for total and differential cell counting. Differential cell counts were performed using Diff Quik (Dade Diagnostics) on aliquots from peritoneal lavage fluid (PLF) samples, counting and identifying a minimum of 350–450 cells/sample.

Detection of IL-4 protein by ELISA. Levels of murine IL-4 within PLF were determined using a standardized sandwich ELISA protocol, with antibodies purchased from PharMingen (San Diego, CA), as previously described (20). In brief, ELISA plates were coated with the rat anti-mouse IL-4 capture antibody (BVD4-1D11) and blocked with BSA, as previously described (20). Following the addition of experimental samples, the biotinylated rat anti-mouse IL-4 detection antibody (BVD6-24G2) was added for 3 h, and streptavidin-alkaline phosphatase (1:20,000 dilution) was used to visualize IL-4. An ELISA amplification system (GIBCO-BRL, Burlington, ON, Canada) was used to increase the color intensity generated, and recombinant murine IL-4 (Intermedico) was used to generate the standard curve.

Histology and quantification of tissue eosinophilia. To assess the tissue inflammatory response to adenoviral infection, intestinal tissues were fixed in 10% neutral buffered formalin overnight, followed by storage in 70% ethanol. Formalin-fixed tissues were then embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin and eosin or hematoxylin and congo red using standard staining procedures. Staining for mucosal and connective tissue mast cells also used standard protocols, as previously described (39). To enumerate eosinophil infiltration, the serosa and muscularis externa of full thickness tissue cross sections were assessed using a Zeiss microscope. Eosinophils were identified as those polymorphonuclear granulocytes with cytoplasms that stained intensely with congo red. The numbers of eosinophils within the serosa and muscularis externa of each cross section were determined, with at least four cross sections studied per mouse, and at least four mice were assessed per group.

Muscle function and carbachol dose-response curves. The preparation of gut sections for muscle contractility analysis has been described previously (39). In brief, the jejunum or ileum was removed and placed in oxygenated (95% O2-5% CO2) Krebs solution, and 1-cm sections of the whole gut were cut from the jejunum, beginning at the ligament of Treitz and proceeding distally, or from the distal ileum, beginning 1 cm from the ileocaecal junction and proceeding proximally. The lumen of each segment was flushed with Krebs buffer prior to the insertion of 2- to 3-mm lengths of Silastic tubing (0.065 outer diameter and 0.030 inner diameter, Dow Corning, Midland, MI) into the open ends of the gut segments, a process that was found to maintain their patency. The tubing was then tied in place with surgical silk. Segments were then hung in the longitudinal axis and attached at one end to a Grass FT03C force transducer (Grass, Quincy, MA), and responses were recorded on a Grass 7D polygraph. Tissues were equilibrated for 30 min at 37°C in oxygenated Krebs solution before the experiments were started. Experiments were then conducted to examine the length-tension characteristics of the muscle before and after infection. Segments were stretched by applying tension equivalent to 0–1,250 mg of weight (a range previously found to be sufficient to determine the maximal responsiveness of both control and inflamed tissues), and contraction was assessed following stimulation with 1 µM carbachol (Sigma Chemical, St. Louis, MO). After each application of tension, the length of the tissue and the contractile response were recorded. To examine neural contributions to contraction, tissues were exposed to the neurotoxin TTX (1 µM) for 15 min to inhibit neural activity. At the end of each experiment, tissues were removed, blotted, and weighed, and the optimal tension (TO) and tissue length that gave the maximum contractile response were used to calculate the cross-sectional area of the tissue. For dose-response studies, the previously identified TO for a tissue was applied first, followed by an exposure of gut segments to noncumulative final bath concentrations of 1 nM–1 mM carbachol by the addition of microliter aliquots to 20-ml baths. After the maximal response to each dose was obtained, tissues were rinsed twice and equilibrated in fresh Krebs solution for 15 min before the next agonist dose was added.

Data presentation and statistical analysis. Responses to carbachol were expressed as milligrams of tension per cross-sectional area as previously described (39). Mean tension was calculated from at least 3 segments/mouse. All results are expressed as means ± SE, with n referring to the number of mice tested. Statistical significance was calculated using the Student’s t-test for comparison of two means or ANOVA for the comparison of three or more means. Multiple comparisons were performed using the Neuman-Keuls multiple-comparison test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Serosal application of adenovectors causes transgene expression in serosal and mesenteric cells. Previous studies have shown that the use of pluronic gel solutions increases gene transfer efficiency as well as antisense oligonucleotide delivery to vascular smooth muscle and endothelial cells (5, 12). As with these earlier studies, we found that the application of the gel alone had no deleterious effects on animal survival, as mice undergoing surgery with or without the subsequent application of the gel to the external surface of the small bowel had similar survival rates (95%). Mice recovered from surgery quickly, and, upon the death of sham-treated mice, the treated small bowel invariably appeared normal except for occasional signs of mild edema (not shown).

Following staining for beta-galactosidase (LACZ), numerous blue, positively stained cells were seen on the surface of the gut (Fig. 1A), with most being in the region of the original gel application. When examined under higher magnification, the majority of the infected cells were identified as serosal cells (Fig. 1B), and this identification was confirmed when cross sections were examined (Fig. 1C). Occasionally other cell types, just beneath the serosa and with the appearance of smooth muscle cells, also stained blue (not shown). In addition, many cells in the intestinal mesentery were also infected (Fig. 1D). To quantify the extent of transgene expression, numbers of LACZ-positive cells per high-power field on the serosal surface of the intestine were enumerated. Strong LACZ expression lasted at least 1 wk, with the numbers of infected cells seen after 1 day of infection (342 ± 35 cells/high-power field) remaining constant until day 7 postinfection (297 ± 46 cells/high-power field). By day 12 postinfection, however, the numbers of positively stained cells had decreased by ~50% to 147 ± 25 cells/high-power field, and, by day 21 postinfection, only a few scattered blue-stained cells (3 ± 1 cells/high-power field) could still be detected.


Figure 1
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  		Fig. 1. Serosal and mesenteric cells express transgenic beta-galactosidase (LACZ). A: macroscopic appearance of the small bowel removed from a mouse infected 3 days previously with AdLACZ virus and subsequently stained for beta-galactosidase activity. Note the blue-stained cells clustered on the surface of the intestine (arrow). B: higher-magnification image (x10) of the same tissue showing numerous blue-stained cells on the surface of the intestine. C and D: cross sections of AdLACZ virus-infected small bowel. Note that serosal cells (C) as well as cells in the intestinal mesentery (D) stained positively for beta-galactosidase enzyme (x200).

 
AdCA-IL-4 infection leads to IL-4 gene expression in infected intestinal tissues. Following the detection of LACZ transgene expression on the serosal gut surface, we next examined whether IL-4 gene expression could be detected in these tissues following AdCAIL-4 infection. IL-4 mRNA expression was semiquantitatively assessed using RT-PCR, with total RNA extracted from preparations of the LMMP (including the serosa) from jejunums of mice infected with either Ad5CAIL-4 or Ad5LACZ viruses. As positive and negative controls, we extracted mRNA from intestinal smooth muscle cultures infected with the same two viruses. One experiment, representative of two separate infections, can be seen in Fig. 2, with the predicted 383-bp sized PCR product for IL-4 detected from the gut tissues of mice receiving Ad5CAIL4 and in cells infected with the IL-4 virus but not in tissues or cells infected with the AdLACZ control vector.


Figure 2
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  		Fig. 2. IL-4 gene transcription in outer layers of the infected small bowel. IL-4 gene transcription was assessed within the serosa and external muscle layers of the small bowel of mice infected 3 days previously with either Ad5CAIL-4 (lane 1) or Ad5LACZ (lane 2) viruses. IL-4 gene transcription was also assessed in intestinal smooth muscle cells in culture that had been infected for 3 days with the Ad5CAIL-4 (lane 3) or Ad5LACZ virus (lane 4). The IL-4 gene transcript was only detected in those tissues and cells infected with the Ad5CAIL4 virus. Hypoxanthine phosphoribosyl transferase (HPRT) was used as the internal housekeeping control in each lane.

 
IL-4 protein levels are dramatically elevated in PLF. To quantitatively assess the local production of IL-4 transgene protein, we assayed PLFs from infected mice by ELISA. Peak IL-4 protein levels within the peritoneal cavity were detected on day 1 postinfection, reaching almost 70 ng of IL-4 protein (~17 ng/ml PLF). Consistent with the observed time course of LACZ transgene expression, high levels of IL-4 protein remained detectable in the PLF for at least 12 days following IL-4 gene transfer (Fig. 3). By day 21 postinfection, levels of IL-4 had become quite low, although still detectable. In contrast, the endogenous levels of IL-4 detected in the PLF from mice receiving the control vector were extremely low, never reaching total levels >2 ng.


Figure 3
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  		Fig. 3. IL-4 protein levels in peritoneal lavage fluid (PLF). The IL-4 protein content per milliliter of PLF taken over the course of the infection from both control- and IL-4 virus-infected mice was assessed by ELISA. IL-4 levels in IL-4 virus-infected mice are represented by filled bars, whereas LACZ virus-infected levels are shown by the open bars, over the course of 3 wk postinfection. Levels of IL-4 protein were significantly higher (*P < 0.01) in AdCAIL-4 virus-infected mice at all time points studied. Results are shown as means ± SE of groups of 4–6 animals.

 
Elevated IL-4 protein expression is localized and not systemic. To determine whether this gene transfer approach had systemic effects, leading to detectable IL-4 expression within the circulation, sera were taken at various time points and assayed for IL-4 by ELISA. Levels of detectable IL-4 within the sera of mice receiving the IL-4 virus were very low (<1 ng) and, similar to the levels found in mice receiving the Ad5LACZ virus, did not go above background (not shown). These results indicate that the delivery and expression of the IL-4 transgene in this model was localized to the peritoneal cavity and did not cause systemic IL-4 elevation. However, other routes of adenoviral infection were capable of elevating IL-4 serum levels, since mice given 2 x 108 pfu of AdCAIL-4 virus by an intramuscular injection were found to have >10 ng IL-4/ml serum on day 3 postinfection.

Adenoviral gene transfer causes a peritoneal inflammatory response. Since IL-4 protein levels were highly elevated in the peritoneal cavity, the impact of adenoviral infection and IL-4 transgene expression was first assessed on the cellular composition within the PLF. To quantify the host inflammatory and immune responses, we performed total and differential cell counts within the PLF. Uninfected mice had <1 million cells in the peritoneal cavity, with most being peritoneal macrophages (75%) and mast cells (15%). Following infection with either adenoviral vector, there was a rapid increase in cell numbers. Peritoneal macrophages, lymphocytes, and neutrophils were increased in number by day 1 postinfection with either virus; however, the expression of IL-4 also recruited eosinophils and mast cells. This trend continued over the following days, with the numbers of inflammatory cells continuing to increase in both groups of animals, although to a greater extent in Ad5CAIL-4 infected mice (see Fig. 4A), and peaked on day 12 postinfection (>180 million cells) compared with a peak on day 7 in AdLACZ-infected mice (45 million). While the cellular response to AdLACZ infection consisted of macrophages, lymphocytes, neutrophils, and a small number of peritoneal mast cells (Fig. 4B), the addition of IL-4 not only increased the numbers of macrophages and lymphocytes over those of the control virus (by ~200%) but also resulted in substantial eosinophilia and plasmacytosis (Fig. 4C). Peritoneal mast cell numbers were also dramatically increased in IL-4 virus-infected mice. By day 21 postinfection, as the infections were clearing, the numbers of inflammatory cells decreased, although they were still elevated 15- and 30-fold over those of control mice. At this time, LACZ- and IL-4 virus-infected mice carried phenotypically similar PLF cell populations except that mast cells and plasma cells were still more numerous within IL-4 virus-infected mice.


Figure 4
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  		Fig. 4. Inflammatory response in PLF. A: total numbers of cells recovered from the PLF taken from both Ad5LACZ and Ad5CAIL-4 virus-infected mice over 3 wk postinfection. Number of cells recovered from Ad5CAIL-4 virus-infected mice are represented by filled bars, whereas numbers from Ad5LACZ virus-infected mice are shown by open bars. The cell phenotypes within the PLF of Ad5LACZ (B) and Ad5CAIL-4 (C) virus-infected mice were subsequently broken down into mast cells (MC), peritoneal macrophages (PM), lymphocytes (LC), eosinophils (EO), neutrophils (NU), and plasma cells (PC). Results are shown as means ± SE of groups of 4–6 animals.

 
IL-4 gene transfer causes tissue inflammation characterized by eosinophilia. Inflammatory responses within the mesentery and intestinal muscularis externa of infected mice were also examined to identify the inflammatory cells that infiltrated these tissues as part of the host response to adenoviral infection and to determine what effect IL-4 overexpression had on cellular phenotypes. Consistent with the PLF findings, the control vector elicited a modest inflammatory response in the mesentery (Fig. 5 A), which peaked on day 7 postinfection and was composed of a mixture of mononuclear cells and neutrophils. Eosinophils were rarely seen in Ad5LACZ-infected mice. A similar histological profile was localized to the serosa and external muscle layers of the jejunum of these mice, with little involvement deeper within the intestinal wall (Fig. 5C). Fewer than 5 eosinophils/intestinal cross section were identified in AdLACZ-infected mice on day 7 postinfection. In contrast, the inflammatory response was stronger in the mesentery (Fig. 5B) and outer muscle layers of AdCAIL-4 infected mice. Significant eosinophilia was observed in the mesentery and muscle layers of mice overexpressing IL-4 (Fig. 5D), along with the infiltrating mononuclear cells and neutrophils seen with the control virus. Congo red staining revealed that eosinophil accumulation began as early as 1 day after IL-4 gene transfer and was marked between days 2 and 12 in the region where the virus was applied. Compared with the virtual absence of eosinophils in control virus-infected tissues, the average tissue infiltrate in the serosa and muscularis externa at day 7 post-AdCAIL-4 infection was composed of 247 ± 56 eosinophils/cross section. This tissue eosinophilia was patchy, however, since some regions showed far more infiltrating eosinophils than others. Despite the eosinophilia seen in IL-4 virus-infected mice, no obvious increase in mucosal mast cell numbers was seen, as assessed by alcian blue staining. While a minor increase in connective tissue mast cell numbers was seen in IL-4 virus-infected mice some 30 days postinfection (using toluidine blue staining), this was also seen in tissues infected with the control virus (not shown).


Figure 5
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  		Fig. 5. Tissue inflammatory response. A: histology (hematoxylin-eosin staining) showing the inflammatory response within the mesentery and on the surface of the intestine of an Ad5LACZ virus-infected mouse at day 7 postinfection (x20 magnification). B: histology of the mesentery and jejunum of an Ad5CAIL-4 virus-infected mouse (also at day 7 postinfection) showing that a stronger inflammatory response developed in these mice. C: congo red staining showing jejunal muscle layers of an Ad5LACZ virus-infected mouse (x200 magnification). Note that eosinophils are absent from the tissue. D: in contrast, congo red staining showed mild tissue eosinophilia in jejunal muscle layers of an IL-4 virus-infected mouse, with the eosinophils designated by arrows (x200 magnification). E: jeunal serosa and muscle layers (x200 magnification) of a pACCMVmIL-5 virus-infected mouse demonstrating significant eosinophilia (see arrows), also at day 7 postinfection.

 
Longitudinal muscle hyperresponsiveness following IL-4 gene transfer. We next examined the contractile response of longitudinal smooth muscle taken from the jejunum and ileum following LACZ and IL-4 gene transfer to the jejunum. No changes in maximum tension generation by longitudinal muscle from either the jejunum or ileum were observed during the course of LACZ infection (Fig. 6, A and B). In contrast, the tension generation by jejunal longitudinal muscle from IL-4 virus-infected mice was modestly increased over control values by day 5 postinfection (not shown), but by day 7 postinfection, the contractile response to carbachol was significantly elevated over the tension generated by both control virus- and AdLACZ virus-infected tissue, at a concentration range of 10–3–10–6 M (Fig. 6A). The neurotoxin TTX had no effect on the muscle hyperresponsiveness, indicating that the changes in function were myogenic and not neural based. While a modest increase was also seen in ileal muscle function (Fig. 6B) following IL-4 gene transfer to the jejunum, it did not reach statistical significance.


Figure 6
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  		Fig. 6. Muscle contractility: jejunal and ileal smooth muscle dose-response curves. Dose-response relationships for carbachol-induced contractions of jejunal (A) and ileal (B) longitudinal smooth muscle are shown. Tissues were taken from control C57BL/6 mice (triangles) or mice infected 7 days earlier with Ad5LACZ (circles) or Ad5CAIL4 (squares) viruses painted on the jejunal surface. *Significant increase (P < 0.01) in tension generation compared with contractions from control and Ad5LACZ virus-infected tissues. Results represent mean tensions ± SE of groups of 4–6 animals.

 
To determine whether the modulation of longitudinal muscle function by IL-4 was specific to the jejunum, a separate group of mice underwent AdCAIL-4 adenoviral application to the serosal ileal surface. After 7 days, muscle contraction was assessed in both the jejunum and ileum. Under these conditions, a significant increase in muscle contraction was identified in both jejunal and ileal longitudinal muscle (not shown), suggesting that the modulatory effects of IL-4 gene transfer on longitudinal muscle are not selective to the jejunum, although this region of the small bowel might be more sensitive to its effects.

IL-5 gene transfer causes tissue eosinophilia but does not alter muscle function. To assess the role of eosinophils in the muscle hyperresponsiveness and to determine whether the induction of longitudinal muscle hypercontractility was specific to IL-4 or instead reflected a generalized response to Th2 cytokines, an adenovirus encoding murine IL-5 was applied to the serosal surface of the mouse small bowel in the same fashion as was used for AdCAIL-4. The resultant overexpression of IL-5 led to heavy tissue eosinophilia in the mesentery and jejunal muscularis externa of infected mice (Fig. 5F), with 449 ± 82 eosinophils/jejunal cross-sectional area found on day 7 postinfection. Despite a tissue eosinophilia even greater than that found following application with the AdCAIL-4 virus, jejunal longitudinal muscle from AdIL-5 virus-infected mice maintained a similar contractility to that exhibited by uninfected or control virus-infected tissues (Fig. 7).


Figure 7
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  		Fig. 7. Infection by Ad5CAIL-4 selectively increases muscle contractility. Shown is the maximum tension generated by jejunal longitudinal muscle in response to 1 µM carbachol from control uninfected C57BL/6 mice (shaded bar) as well as mice given the Ad5LACZ marker virus (open bar), Ad5CAIL-4 virus (filled bar), or pACCMVmIL-5 (spotted bar). Results are means ± SE of groups of 4–6 animals. *Significant increase (P < 0.01) in tension generation compared with the other 3 groups on day 7 postinfection.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Significant interest has been expressed in the potential use of viral vectors for the immunotherapy of a variety of GI diseases (20, 24, 31). In addition, we and others have proposed using these vectors as tools to elucidate the pathology and pathophysiology of inflammatory conditions of the GI tract (42, 43, 50). This study provides the first demonstration of successful, site-directed gene transfer to the serosal surface of the murine small bowel and also demonstrates that IL-4 plays a causal role in the induction of smooth muscle hyperresponsiveness in the GI tract. Previous studies have implicated IL-4 and other Th2 cytokines in the alteration of intestinal smooth muscle function during nematode infections (3, 15); however, it was unclear whether this immunomodulation was a selective feature of parasite infections or, alternatively, reflected a generalized response of the enteric neuromuscular apparatus to both infectious and idiopathic-based inflammation. In fact, alterations in smooth muscle physiology are thought to contribute to the disease process and symptomatology of a number of inflammatory diseases including the bronchoconstriction seen in asthma (30) and the pain, nausea, and diarrhea that occur in IBD (9). Why smooth muscle dysfunction occurs concomitantly with mucosal inflammation is currently unclear; however, increased muscle contraction in the small bowel of rodents also occurs during enteric infection by nematode parasites (9, 38, 44), bacteria (33), and protozoa (10). We and others have postulated that these motility disturbances are an adaptive host response, resulting from the immune system recruiting the gut neuromuscular apparatus to aid in the expulsion of pathogens through increased propulsive activity (9, 13, 38).

Using nematode infection models to address the mechanisms involved in muscle hyperresponsiveness, a causal role was initially identified for T lymphocytes and their mediators, since muscle hypercontractility was attenuated in T. spiralis-infected mice lacking T lymphocyte function (40, 41, 45). Considering that nematode infections are associated with predominantly Th2 immune responses (13), our research later focused on the actions of eosinophils and mast cells as well as the Th2 cytokines IL-5, IL-13, and IL-4, which are upregulated during T. spiralis infection. Additional studies determined that the development of increased muscle contractility was significantly attenuated in T. spiralis-infected IL-4 deficient mice and in STAT6-deficient mice (22). While these studies suggested that IL-4 contributed to muscle dysfunction during parasite infections, its role in other, nonparasite-induced forms of intestinal inflammation was unclear. Overexpression of IL-4 in proximity to the gut neuromuscular apparatus appeared to be the most direct approach to address this issue. Although targeting the transgene to the intestinal mucosa might better mimic the pathological conditions associated with altered GI motility, the difficulties encountered targeting gene transfer through the intestinal lumen are numerous. Beyond the protective epithelial and mucus barriers that line the lumen, limiting viral infection, a previous study found that delivery of the IL-4 virus to the colonic lumen caused a lethal colitis in most mice (43). Moreover, because of epithelial cell turnover (8), transgene expression lasted only 3 days. We circumvented these problems by bypassing the intestinal lumen and targeting the outer layers of the gut using a biocompatible pluronic gel. Thermally reversible gels have been used in a number of studies, including gene transfer (12), and, because of their low toxicity and lack of irritancy, they have also been investigated as drug delivery systems for opthalmic, rectal, and subcutaneous use (28, 29). Gel application, in this study, allowed for the targeting of specific regions of the intestine and enabled significant transgene expression, presumably by increasing the duration of the vector’s bioavailability.

Our results demonstrate that localized overexpression of transgenic IL-4 was sufficient, in the context of an adenoviral infection, to induce longitudinal muscle hypercontractility in mice. It is conceivable that IL-4 exerted this modulatory effect indirectly, through the actions of inflammatory cells, since the adenoviral infection itself led to an inflammatory response centered on the intestinal serosa and mesentery. Moreover, the IL-4 transgene expression altered the host response to one typical of allergic inflammation, including significant tissue eosinophilia. Eosinophils produce mediators that can contract smooth muscle in vitro and are frequently associated with smooth muscle hyperresponsiveness (23, 30). We previously found that T. spiralis-induced eosinophilia was attenuated in IL-5-deficient mice, concurrent with a modestly attenuated muscle dysfunction (37). However, in the present study, although infection with an IL-5-encoding adenovirus caused local eosinophilia, it did not cause altered muscle function; therefore, evidence of a direct role for eosinophils in inducing the muscle hypercontractility is lacking. Mast cells are another cell type frequently linked to smooth muscle dysfunction; however, earlier studies assessing the role of mast cells found that following T. spiralis infection, mast cell-deficient WWv mice still developed significant muscle hypercontractility (39). Thus, the mere presence of eosinophils and mast cells within affected tissues appears insufficient to alter muscle function.

Interestingly, IL-4 overexpression can have quite different effects on the gut neuromuscular apparatus, depending on the context. Finkelman and colleagues (13, 14) found that mice injected with large doses of a long-lasting IL-4 protein complexed to neutralizing anti-IL-4 antibodies developed a massive increase in mucosal mast cell numbers within the small bowel. Furthermore, Goldhill et al. (15) found this treatment was associated with a significant increase in the electrical field-stimulated response of longitudinal smooth muscle that was mediated by mast cells and leukotrienes, as assessed by the use of WWv and 5-lipoxygenase-deficient mice (15). In contrast, our increased muscle contractility was not dependent on enteric nerves, since it was TTX independent. Moreover, our treatment did not cause an increase in mucosal or connective tissue mast cells within intestinal tissue. The basis for this difference might be the systemic nature of the IL-4 treatment in Finkelman et al.’s work, since the injected IL-4 complexes affected the bone marrow, increasing the number of mast cell precursors (13, 14), whereas in our approach, IL-4 elevation was localized and not systemic. While our two approaches to IL-4 overexpression affected different aspects of intestinal motility, both forms of immunomodulation may well occur during enteric infections, providing complementary enhancements of gut neuromuscular function.

With our inability in this and previous studies to directly link intestinal smooth muscle hyperresponsiveness to the actions of eosinophils or mast cells, another possibility is that locally expressed IL-4 acts directly on the smooth muscle itself. Preliminary studies have identified strong mRNA expression of the {alpha}-chain of the IL-4 receptor in murine jejunal LMMP preparations, whereas cultured intestinal smooth muscle cells also express this receptor (2, 4). Moreover, studies of isolated murine and human intestinal smooth muscle cells found that preincubation of these cells with IL-4 significantly enhanced the strength of their carbachol-induced contractions (2, 4). Whether this effect happens in vivo and if it requires a concurrent inflammatory response are important questions to address in the future, as is the perceived sensitivity of jejunal muscle to IL-4’s immunomodulatory effects. Jejunal sensitivity might reflect a predetermined stomach to a cecum gradient in altered muscle function within the intestine, as a means to promote pathogen expulsion from the GI tract. Additional studies are also needed to address the role of IL-4 in the modulation of smooth muscle function in other more clinically important forms of intestinal inflammation. For example, Crohn’s disease frequently leads to transmural involvement of affected intestinal segments (11), and, interestingly, despite the predominantly T helper type 1 (Th1) immune response found in the inflamed mucosa, recent studies have found elevated IL-4 protein levels in the muscularis externa of inflamed tissue specimens from Crohn’s disease patients (4). Future studies will need to determine the cellular source of the IL-4 as well as the basis for its expression in the face of a Th1 mucosal immune response.

In summary, this study demonstrates the utility of targeting the outer layers of the gut for intestinal gene transfer compared with luminal approaches. This approach led to efficient targeting of the intestinal surface with transgene expression lasting at least 2 wk. While our study required surgery, intestinal serosal cells and cells in the mesentery are potentially the most accessible cell types to target for transfection through laparoscopic approaches. Moreover, this study demonstrates that transgene-based overexpression of IL-4 had dramatic effects on the function of the enteric smooth muscle. These and previous studies have provided strong evidence that the gut motor system is subject to functional modulation by the host immune system during parasitic and now viral infections. While this recruitment of the gut motor system may be a form of innate host defense against enteric pathogens, understanding these processes may also prove relevant to asthma and IBD, since these conditions are thought to be caused by the immune system inappropriately perceiving environmental antigens as threats and responding to them as such. Therefore, studying the interactions between the immune and intestinal neuromuscular systems may prove useful not only in understanding how these two systems participate in host defense (9) but also in the development of therapies to attenuate these maladaptive responses during idiopathic GI diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by operating grants from the Canadian Institutes of Health Research (to S. M. Collins and B. A. Vallance). B. A. Vallance is the Children with Intestinal and Liver Disorders Foundation Research Scholar, a Michael Smith Foundation for Health Research Scholar, and the Canada Research Chair in Pediatric Gastroenterology.


   ACKNOWLEDGMENTS
 
We thank Patricia Blennerhassett for technical assistance and Ruth Cheung for administrative assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. A. Vallance, British Columbia’s Children’s Hospital, ACB, Rm. K4-188, 4480 Oak St., Vancouver, BC, Canada V6H 3V4 (e-mail: bvallance{at}cw.bc.ca)

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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