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Am J Physiol Gastrointest Liver Physiol 293: G383-G390, 2007. First published April 12, 2007; doi:10.1152/ajpgi.00341.2006
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INNOVATIVE METHODOLOGY

Eosinophilia is induced in the colon of Th2-sensitized mice upon exposure to locally expressed antigen

Qing Zhu,1,* Christopher W. Thomson,1,* Guofeng Zhang,3 Martin Stämpfli,1 Mark R. McDermott,1 Stephen M. Collins,2 and Jack Gauldie1

Departments of 1Pathology and Molecular Medicine, Center for Gene Therapeutics and 2Medicine, McMaster University, Hamilton, Ontario, Canada; and 3Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland

Submitted 26 July 2006 ; accepted in final form 23 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Eosinophilic inflammation is a feature of a variety of gastrointestinal (GI) disorders including eosinophil-associated GI disorder, allergy, inflammatory bowel disease, and parasite infection. Elucidating the mechanisms of eosinophil infiltration into the GI tract is important to the understanding of multiple disease processes. We hypothesize that eosinophilia in the large intestine (colon) can be induced by an antigen in a host that is associated with Th2-skewed antigen-specific immune responses. To investigate the importance of antigenic triggering, we established polarized antigen-specific Th2 type responses in BALB/c mice, using ovalbumin in conjunction with aluminum hydroxide. Upon challenge at the colonic mucosa through transient (3–4 days) expression of the antigen gene encoded in an adenovirus vector, sensitized animals developed significant subepithelial colonic inflammation, characterized by marked eosinophilic infiltration, and the presence of enlarged and increased numbers of lymphoid follicles. The alterations peaked around day 5 and resolved over the next 5–10 days, and no epithelial cell damage was detected through the entire course. Administration of a control (empty) adenovirus vector did not lead to any pathological changes. These data suggest that colonic eosinophilia can be induced by exposure to an antigen associated with preexisting Th2-skewed responses. Thus the model established here may provide a useful tool to study GI and, in particular, colonic inflammation with respect to underlying mechanisms involved in the recruitment and the immediate function of eosinophils.

mucosal; eosinophil; adenovirus; intrarectal

EOSINOPHIL ACCUMULATION IS a hallmark of allergic disease orchestrated by Th2-type cytokines (16). In addition to participating in pulmonary allergic diseases (e.g., asthma), eosinophils as resident and recruitable cells in the gastrointestinal (GI) mucosae play a critical role in responses to parasitic infection as well as in the regulation of gastrointestinal allergy (9, 45). Eosinophil accumulation is a profound cellular inflammation, often seen as a component of eosinophil-associated GI disorders (EGIDs). EGIDs involve most parts of the GI tract, including disorders such as eosinophilic esophagitis, eosinophilic gastritis, and gastroenteritis. This series of disorders can affect people of all ages, from infants through the elderly (27), and is commonly seen in food allergy, allergic colitis, and inflammatory bowel disease (Crohn's disease and ulcerative colitis) (5, 43, 46).

Although the underlying mechanisms involved in the development of GI eosinophilia still remains elusive, studies have shown that eosinophils are mainly recruited from the bone marrow through the blood circulation, with recruitment pivotally regulated by IL-5, a factor that is crucial in eosinophil generation, differentiation, and activation (6, 30, 46). Infiltration of these cells into local tissues is, however, regulated primarily by the chemokine eotaxin (12, 14, 35, 37). Eosinophil presence is believed to contribute directly to GI disease pathogenesis, leading to tissue destruction and clinical symptoms such as diarrhea, vomiting, and mucosal bleeding (40, 49). Some studies have demonstrated that eosinophil degranulation correlates with the severity of gastroenteritis (6, 52) and eosinophil infiltration into the mucosal lamina propria during active colitis is thought to contribute to the pathogenesis of the disease (10, 53).

It has been previously shown that when a host acquires a Th2-type immune response to a specific antigen, airway inflammation with marked Th2-like eosinophilia can be induced after aerosol challenge with the sensitizing antigen (41). In a limited number of murine models, it has been demonstrated that significant eosinophilic inflammation can occur in the upper GI tract (esophagus and stomach) and small intestines (23, 33, 34). Here we develop a mouse model of colonic eosinophilia to address whether similar eosinophilic inflammation can be induced in the large intestine associated with the Th2 response. The model reveals that mice sensitized with intraperitoneal (IP) injection of ovalbumin antigen (OVA) adsorbed to aluminum hydroxide mount Th2 type immune responses against the antigen both systemically and locally in the colon. After OVA challenge through adenovirus vector transient gene transfer (3–5 days of expression) of OVA to the rectal mucosal epithelium, sensitized but not naive mice developed notable inflammation in the colon, which peaked at day 5, and large lymphocytic follicles within the subepithelial regions of the colon significantly increased in number and size. Moreover, substantial eosinophilic inflammation was identified in the colon lamina propria. Eosinophilia was transient in this model and did not cause significant epithelial destruction. There was a decrease in the tissue inflammatory response over time and a return to normal structure and function of the colon by 15 days after antigen challenge. This study provides a new understanding of the influence of preexisting antigen-specific cytokine imbalance on antigenic triggering of the large intestine mucosal immune system to develop eosinophilic inflammation and demonstrates the potential reversibility of immunopathological changes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and cell lines. Female BALB/c (H-2d) mice aged 6–8 wk were purchased from Charles River Laboratories (Troy, NY) and housed in pathogen-free conditions at Central Animal Facility at McMaster University. All animal experiments were approved by the Animal Ethics Research Board of McMaster University and conducted according to regulations of the Canadian Council on Animal Care.

Adenovirus vectors. AdLuc is a replication-deficient adenovirus vector that expresses firefly luciferase (1). AdOVA is an adenovirus vector that expresses chicken OVA (Zhu Q, Thomson CW, Rosenthal K, McDermott MR, Collins SM, and Gauldie J, unpublished observations). Adenovirus vector were propagated in 293 cells and purified by cesium chloride gradient centrifugation (22). All virus stocks were aliquoted and stored at –70°C before use.

Th2 precondition and antigen challenge. As described in previous studies (41), mice were sensitized to OVA by 2 IP injections of 8 or 100 µg OVA (Grade V, Sigma-Aldrich, Oakville, ON, Canada) absorbed to 4 mg aluminum hydroxide [Al(OH)3; Sigma-Aldrich] in PBS given 5 days apart. Seven days after the second sensitization, mice were challenged with AdOVA either intranasally (IN) or intrarectally (IR). Tissues were collected at days 5, 10, 15, and 20 postchallenge for histological analysis.

ELISA. ELISA was performed as previously described (4, 13). Briefly, sera, lung, or colorectal homogenates were serially diluted and incubated in OVA protein (Grade V, Sigma-Aldrich, St. Louis, MO) precoated plates for 2 h. The plates were then incubated with alkaline phosphatase-conjugated anti-mouse IgG1 or IgG2a detection antibodies (Southern Biotechnology Associates, Birmingham, AL) for 2 h and color was developed by incubating p-nitrophenyl phosphate for 30 min in darkness. The optical density was read at 405 nm on TECAN (Research Triangle Park, NC). Antibody titers were derived from the inverse dilution at which the sample yielded an optical density twice that of the background of control specimens from nonimmunized mice.

Luciferase assay. Luciferase activity was measured by using a luciferase assay system kit (Promega, Madison, WI) according to manufacturer's instructions. Briefly, colons were homogenized with a homogenizer (POLYTRON, Kinematica, Cincinnati, OH) and placed in cell culture lysis reagent. After centrifugation, 20 µl of supernatant were plated on a LumiNunc MicroWell plate (Nalge Nunc, Rochester, NY) and assayed on a Tropix TR-717 microplate luminometer (Applied Biosystems, Bedford, MA) following the addition of 100 µl of 5 µg luciferase assay reagent (beetle luciferin) to each well and immediate measurement of emitted light. The enzyme activity was determined by an increase in relative light units per tissue weight compared with background measured in untreated mice.

BAL. Lung was lavaged with three aliquots of PBS (200 µl) to recover bronchoalveolar lavage (BAL) fluid. Total cell counts were determined with a hemocytometer. Cells were pelleted and resuspended in PBS and used to prepare smears by cytocentrifugation (Shandon, Pittsburgh, PA) at 300 rpm for 2 min. All smears were stained with Diff-Quik stain (Baxter, McGraw Park, IL). Differential cell counts in BAL were determined from at least 500 leukocytes by using standard hemocytological criteria to classify the cells.

Microscopy. Colons were removed and flushed of luminal contents with PBS. Lungs were removed after the BAL procedure. After 24-h fixation in 10% formalin, tissues were paraffin embedded, sectioned at 4–5 µm, and stained with Mayer's hematoxylin and eosin. To identify eosinophils, Congo red and Mayer's hematoxylin were used (51). Digital images were captured using the Leica Q500IW image processing and analysis system (Wetzlar, Germany). For transmission electron microscopy, removed colons were prepared as previously described (56). In brief, colon tissues were fixed in 2.5% of paraformaldehyde and 2.0% of glutaraldehyde. Ultrathin sections were cut, counterstained, and examined for eosinophil activation under a transmission electron microscopy (FEI CM120, FEI).

Morphometric analysis of colonic lymphoid nodules. In addition to counting the number of lymphoid nodules in each specimen, the total area of the lymphoid nodules was calculated using a Leica Q500IW image processing and analysis system and Leica Qwin Pro version 2.3 software. After the microscope was adjusted for optimal viewing at x50, the system was calibrated using a 100-µm ruler to 2.82 µm/pixel. After calibration, all measurements were taken with extreme caution not to alter the viewing area settings.

Statistical analysis. Comparisons between groups were analyzed by Student's t-test. Comparisons among means of more than two groups were determined by one-way ANOVA post hoc with Bonferroni correction. Analyses were performed with SPSS 11.5 for Windows (SPSS, Chicago, IL). P values less than 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of Th2 sensitization and antigen challenge using adenovirus vector. Sensitization of BALB/c mice with 2 IP injections of OVA protein absorbed to Al(OH)3 as described previously (41) led to the development of an antigen-specific Th2-type response to OVA. The titers of OVA-specific IgG1, but not IgG2a, antibodies were found to be increased not only in the serum, but also in lavage fluid recovered from the lung (Fig. 1A). To verify that antigen expression from the colonic epithelial cells through gene transfer by adenovirus vectors can induce inflammatory responses in the lung similar to those induced by aerosolization of OVA protein as described previously (41), 5 x 108 PFU of adenovirus vector expressing OVA (AdOVA) were given by IN route once, 7 days after sensitization. We found that IN AdOVA challenge produced profound pulmonary inflammation 5 days after instillation (Fig. 1B). Compared with histological lung sections taken from sensitized mice challenged with AdBHG (a control adenovirus vector without heterologous gene), which were identical to those from naive mice (Fig. 1B, left), AdOVA-transfected lungs of sensitized mice showed a marked immune response, characterized by lymphocytic infiltration surrounding the pulmonary blood vessels and bronchi (Fig. 1B, right). Eosinophils were rarely seen in control lungs (Fig. 1B, left) but were readily visualized by Congo red stain in AdOVA challenged lungs (Fig. 1B, right). Lymphocytosis and increased eosinophil counts were also observed in the BAL fluid taken from these AdOVA-challenged mice (data not shown). Thus IN antigen challenge of Th2-sensitized mice using adenovirus vectors specifically induced antigen-specific inflammatory responses in a similar manner to repeated antigen aerosolization to the upper airway as previously described (41).


Figure 1
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  		Fig. 1. Establishment of antigen-specific Th2 type sensitization colon model and using adenovirus for antigen challenge. A: BALB/c mice were treated with 8 or 100 µg ovalbumin (OVA) plus 4 mg aluminum hydroxide [Al(OH)3] by 2 intraperitoneal (IP) injections given 5 days apart. Seven days after the second injection, mice were challenged intranasally (IN; for serum and lung) or intrarectally (IR; for colon) and 5 days later ELISA was used to determine IgG1 and IgG2a titers in sera as well as in lung and colon homogenates. The Th2 type response was found to be dependent on antigen dose and detected both systemically and locally in the lung and colon. Results represent means ± SD from 3 mice each group. B and C: inflammatory response in the lung of sensitized mice 5 days after IN challenge with antigen-expressing Ad. Animals sensitized with 8 µg OVA and 4 mg Al(OH)3 by 2 IP injections. Seven days after the second injection, mice were challenged IN with 5x108 PFU of AdOVA or AdBHG (a control Ad without heterologous genes). Five days later, lungs were paraffin-embedded, sectioned, and stained and were examined with a light microscope. B: sensitized mice were challenged IN with AdBHG (left) or AdOVA (right). Lung sections were stained with hematoxylin and eosin (H&E; magnification of x50 in diameter). C: sensitized mice were challenged IN with AdBHG (left) or AdOVA (right) and lungs were stained with Congo red/hematoxylin for eosinophils (a magnification of x400 in diameter). AdOVA-infected lungs showed extensive peribronchial and perivascular inflammations (A, right) and a typical appearance of eosinophilia (B, right; eosinophils in brownish color). Results represent 1 of 5 independent experiments.

 
Colonic Th2 precondition and adenovirus-mediated gene transfer to the colon. We next investigated whether mice sensitized with IP injections of OVA and Al(OH)3 had a Th2-type response in the colon and, if so, whether Th2 sensitization would cause differences in quantity of gene transfer to the colon mucosa compared with naive controls. Similar to observations in the lung, levels of OVA-specific IgG1 antibodies in colonic tissue increased after sensitization, whereas IgG2a antibodies were undetectable (Fig. 1). This suggests that, similar to the lung, the colonic mucosal immune system can also be preconditioned to develop Th2-type responses specific for the antigen after sensitization by IP route.

To investigate gene transfer to the colon, 1x109 PFU of adenovirus vector expressing luciferase (AdLuc) were given by the IR route, 7 days after Th2 sensitization. At days 2, 4, 6, and 8 after vector challenge, entire colons were removed and cut into distal, middle, and proximal sections of equal length for luciferase measurement. Significant levels of luciferase expression were detected and were shown to persist for ~6–7 days (Fig. 2). However, no differences in luciferase gene expression were found between sensitized and naive mice (Fig. 2), suggesting that Th2 sensitization does not lead to alterations in gene transfer ability or modulation of gene expression systems at the colonic mucosa.


Figure 2
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  		Fig. 2. Gene transfer by adenovirus vector to the colon of sensitized mice. Animals were sensitized with 8 µg OVA absorbed to Al(OH)3 by 2 IP injections. Seven days after the second injection, 109 PFU of AdLuc was given by IR. Colons were removed at days 2, 4, 6, and 8, and segmented into 3 equal-length pieces (distal, middle, and proximal) and homogenized. Luciferase activities were measured in each homogenate. Gene transfer in sensitized mice was not statistically different from that in naive mice with respect to the level of gene expression and the length of time. Results are expressed as mean ± SD from 3 mice each group.

 
Induction of colonic inflammatory response after antigen challenge at the colonic mucosa. Eosinophilia of the upper GI tract and small intestine can be experimentally induced in mice through oral feeding of allergen (23, 34). We here examined whether colonic eosinophilia can be induced in these Th2-sensitized mice upon exposure to the OVA antigen at the mucosa of the large intestine. Seven days after sensitization as described above, mice were challenged IR with 1x109 PFU of AdOVA and colons were removed for examination of histological changes at various time points. In AdBHG (control vector)-challenged mice, there were only one or two visible lymphoid aggregates, relatively small in size, and indistinguishable from those seen in naive animals (Fig. 3A). However, in AdOVA-challenged mice, on gross examination, we observed some degree of redness, edema, and hypotonia of the colon and the presence of vascular ectasias in the lining and wall of the colon (Fig. 3B). Histological sections of the colons of these mice showed hyperplasia of the lymphoid follicles with lymphocytic infiltration, across the lamina propria and submucosa (Fig. 3B). Enlarged lymphoid follicles were increasingly dispersed along the entire length of the colon (Fig. 3C, part of colon shown).


Figure 3
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  		Fig. 3. Inflammatory responses in the colon of sensitized mice 5 days after antigen challenge with adenovirus vector by IR route. Seven days after OVA (8 µg) sensitization, mice were challenged IR with 109 PFU of AdOVA or AdBHG. At day 5 after IR challenge, animals were killed and colons were paraffin embedded, sectioned, and stained with H&E and were examined with a light microscope. A: gross examination (top) and colon section (bottom) from sensitized mice challenged with AdBHG. B: gross examination (top) and colon sections (bottom) from sensitized mice challenged with AdOVA. C: part of colon is shown for distribution of lymphoid follicles. In AdBHG-infected mice, there were only 1 or 2 visible lymphoid nodules, relatively small in size, and indistinguishable from those seen in naive animals. In AdOVA-infected mice, lymphocytes infiltrated the colon. The lymphoid follicles were enlarged and more than 2 follicles found. Results represent 1 of 5 independent experiments. Magnification of x50 in diameter.

 
Further analysis was performed to assess the histological changes in the colon, including morphometric analysis of colonic lymphoid nodules using the Leica Q500IW image processing and analysis system and Leica Qwin Pro version 2.3 software. All lymphoid nodules were counted under light microscopy and the total cross section of full-length colon area was measured on one slide from each mouse under the same instrument settings. Numbers of lymphoid nodules and total areas were compared between naive/no challenge, sensitized/AdBHG challenge, and sensitized/AdOVA challenge groups. It was found that AdOVA challenge resulted in a two- to threefold increase of both parameters measured, whereas challenge with the AdBHG control vector did not have significant impact on histological changes compared with the naive control (Fig. 4).


Figure 4
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  		Fig. 4. Increased number and total area of lymphoid nodules after antigen challenge with adenovirus vector by IR in sensitized mice. OVA (8 µg)-sensitized mice were challenged with AdOVA or AdBHG 7 days after sensitization. Five days later, colons were removed and analyzed under a microscope. A: number of visible lymphoid nodules were counted on one histological section from each group. Increasing number of lymphoid nodules was observed after AdOVA IR challenge compared with controls (**P < 0.01, n = 5/group). B: cross-sectional area of all lymphoid nodules (total area) were analyzed morphometrically by using image analysis software. There was an increase in total area of lymphoid nodules after AdOVA IR challenge (**P < 0.01, n = 5/group).

 
Eosinophilic infiltration was examined by using Congo red stain on colon sections obtained from challenged mice. Challenge of sensitized mice with the control vector AdBHG by the IR route did not induce a significant appearance of eosinophils in the colon (Fig. 5A). However, upon IR challenge with AdOVA, a large number of eosinophils was present in the colonic lamina propria at day 5 postinfection (Fig. 5B). This suggests that colonic eosinophilia can be induced in association with local antigen exposure as seen in the lung in Th2-sensitized animals.


Figure 5
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  		Fig. 5. Colonic eosinophilia after antigen challenge with adenovirus vector in sensitized mice. Animals sensitized to OVA were challenged IR with 109 PFU of AdOVA or AdBHG 7 days after sensitization. Five days later, Congo red/hematoxylin stain for eosinophils was applied on paraffin-embedded sections of the colon. A: sensitized animals were challenged IR with AdBHG. Eosinophils were rarely found in the colon. B: sensitized animals were challenged with AdOVA. A significant eosinophilia was detected in the lamina propria. Results represent 1 of 5 independent experiments. A magnification of 400 in diameter.

 
The colonic inflammatory response was also examined in sensitized mice at days 10, 15, and 20 postchallenge with AdOVA. Analysis of these colonic tissues with respect to lymphoid follicles and eosinophils revealed that at these later time points inflammation was markedly decreased. There were no overt histological changes at day 15 after challenge compared with controls, suggesting that inflammation began to resolve by day 10–15.

Eosinophils are involved in the inflammatory response by discharging their granule-specific proteins at site of inflammation (45). We thus examined colon sections by transmission electron microscopy and asked whether infiltrated eosinophils are activated in the colonic mucosa after antigen challenge. Compared with naive (Fig. 6A) and sensitized animals with irrelevant antigen challenge (Fig. 6B), colonic eosinophils of OVA-challenged mice displayed a moderate degree of piecemeal degranulation, which is indicated by increased numbers of granules with reduced electron translucent matrix density or halos (Fig. 6C). Although these intracellular granules were significantly increased in number than those of controls (Fig. 6D), we did not observe extensive degranulation such as loss of electron dense core or morphologically altered granules.


Figure 6
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  		Fig. 6. Granules in colonic eosinophils. Representative electron microscopy sections of naive mice (A), sensitized mice 5 days after IR challenge with AdBHG (B), or AdOVA (C), are shown. D: eosinophil granules with reduced electron translucent matrix density (black arrow) or halos (white arrow) were calculated as percentage of total granules counted. Results are expressed as mean ± SD from 5 mice each group. The magnification of the photomicrographs presented is x9,300.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previous studies imply that food allergy contributes to the development of GI eosinophilia (36, 48). Eosinophil infiltration can be triggered by hypersensitivity to cow's milk, and the tissue eosinophilia decreases on withdrawal of milk from the diet (21, 28, 32, 48) and Th2, but not Th1, cytokine profiles were found in patients with milk hypersensitivity (3). Rothenburg and colleagues (23, 33, 34) developed a murine model of eosinophil-associated allergy, in which mice sensitized with OVA and aluminum hydroxide are fed with the same antigen by oral route. They identified eosinophils in the upper GI mucosa and related the findings to the presence of Th2-type cytokines and specific chemokines such as eotaxin (23, 24). Naturally, the presence of luminal bacteria probably drives immune responses toward either a Th1 or Th2 cytokine bias, and such cytokine imbalance might promote the disease (19, 20). The recognition of microbial components by intestinal epithelial cells through Toll-like receptors may play an important role in establishing the polarized cytokine profile (26), and CD4+ T cells may be essential in producing these cytokines (2, 25, 58). We here demonstrate that Th2-sensitized mice, but not naive mice, could develop colonic eosinophilic inflammation after antigen challenge by the rectal route. Therefore, preexisting biased Th2 immune response and expression of associated antigen appear responsible for the development of colonic eosinophilic inflammation and may also apply to a number of human GI conditions.

Studies (11, 55; Zhu Q, Thomson CW, Rosenthal K, McDermott MR, Collins SM, and Gauldie J, unpublished observation) have shown that adenovirus vector transfer of antigen genes to the colon results in efficient cell-associated gene expression in the epithelial layer with some expression extending to the lamina propria. In this regard, adenovirus vector proved to be a useful tool for antigen transfer and expression at the lower GI mucosa. In the present study, we challenged mice sensitized to OVA with adenovirus vector encoding the OVA gene by the intrarectal route and showed that antigen transfer and expression induced significant lymphocytic and eosinophilic infiltration, whereas an identical vector that lacks OVA gene (AdBHG) had no appreciable histological effects. Thus antigen expression in the large intestine mucosa of Th2-sensitized mice induces colonic inflammation. We found that adenovirus vector-transferred gene product was expressed and detectable for up to 1 wk in the colon. Peak antigen expression was detected during the initial 1–3 days after IR administration, after which the levels reduced to baseline by 1 wk. The presence of eosinophilic inflammation is dependent on the presence of antigen as the inflammation subsided coincident with the decreased antigen presence in the colon. Antigen presence triggers colonic inflammation and, very likely, is responsible for the entire course of pathological alterations.

Eosinophils can be activated to release a series of cytotoxic granule proteins causing tissue damage, and accumulation of these eosinophils is thought to be detrimental (15, 54) and to be central to the pathogenesis of colitis (10, 39). We did not see the profound inflammation sustained for more than 1 wk nor the development of colonic ulceration, significant weight loss, or diarrhea, although granules with electron translucent matrix were significantly increased in the infiltrated eosinophils. However, no extensive eosinophil degranulation was found. These data suggest that induced eosinophils in the colon alone do not progress to tissue damage and require at least a "second" signal for activation/degranulation. Moreover, the pathological alterations (eosinophilia) are reversible and do not lead to prolonged tissue destruction or remodeling.

Several factors might be contributory to the results. First, exposure to only one antigen might be not sufficient to induce activation or degranulation. An early observation in human disease revealed that sequential dietary eliminations reduced clinical symptom each time (31), implying that multiple allergens are involved to cause disease. Second, the antigen is only present for up to 1 wk after challenge and this might be not long enough to promote disease development (ulceration). Third, the route of challenge might result in different pathological changes. It was shown that challenge of systemic sensitized mice by the intragastric route induced diarrhea, which was accompanied by a dramatic infiltration of eosinophils, mast cells, and CD4+ Th2 cells into the large but not the small intestine (29). It remains to be determined whether these colonic eosinophils can generate severe or irreversible pathological changes upon a more chronic antigen exposure. Because the efficacy of gene expression on repeated adenovirus gene transfer is dramatically reduced owing to the immunogenicity of the vector, different serotypes of adenovirus or other types of antigen-delivery vehicles may be used for chronic antigen challenge. Also, it warrants investigation whether these cells participate only in antigen presentation, which can potentially promote local inflammation and destroy mucosal cells as shown in the respiratory system (50).

In addition to colonic eosinophilia, we also identified lymphoid hyperplasia and the development of multiple lymphoid nodules in the colon on antigen expression. In the large intestine, M cells overlay aggregated lymphoid follicles (42, 44) and smaller isolated lymphoid follicles (17). This aggravated lymphoid response is reminiscent of previously found lymphoid hyperplasia in colitis in mice (7, 8) and humans (18, 57). It is possible that these mucosal lymphoid structures are involved in the development of colonic inflammation. It has been recently shown in the lung that mice lacking secondary lymphoid organs can generate mucosal lymphoid aggregates, called inducible bronchus-associated lymphoid tissue, beneath the epithelium at the branches of the bronchi, allowing the development of protective local immunity upon influenza infection (38). Adoptively transferred CD4+ T cells can undergo clonal expansion in the lamina propria and the epithelial layer of both small and large intestines (47). Thus scattered lymphocytes might also be able to expand in the lamina propria after antigen challenge, leading to a significant number of lymphoid follicles in the mucosa.

In conclusion, Th2-sensitized mice challenged with antigen via the rectal route can induce colonic inflammatory responses, featured by significant eosinophilic infiltration and lymphoid hyperplasia in the lamina propria, which is similar to human EGID. This murine model might provide a new understanding concerning the influence of a preexisting antigen-specific Th2-biased immunological condition on subsequent antigen exposure causing eosinophilic inflammation in the large intestine mucosa and also indicates the potential for reversal of inflammation (eosinophilia) without progression to overt tissue damage.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by a grant from the Canadian Institutes for Health Research.


   ACKNOWLEDGMENTS
 
The authors thank Duncan Chong and Xueya Feng for technical assistance in adenovirus preparation, and Bryan Hewlett and Mary Jo Smith for assistance with immunohistochemical procedures. The authors also thank Pengchang Yang for helpful suggestions and comments on electron microscopy, and Jay Berzofsky and Richard Leapman for enthusiastic support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Q. Zhu, Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (e-mail: zhuq{at}mail.nih.gov)

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.

* Qing Zhu and Christopher W. Thomson contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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