1Department of Internal Medicine, School of Medicine, Keio University, Tokyo, 160-8582; and 2Second Department of Internal Medicine, National Defense Medical College, Saitama, 359-8513, Japan
Submitted 21 April 2003 ; accepted in final form 10 December 2003
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ABSTRACT |
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Brown Norway rats; rat mast cell protease II; delayed type hypersensitivity; Peyer's patch; intercellular adhesion molecule-1
Expression of adhesion molecules is enhanced in inflammatory mucosa, resulting in the migration of inflammatory cells to this lesion. Studies of other atopic diseases and animal models suggest that adhesion molecules and mucosal lymphocytes are implicated in the pathogenesis of food allergy (12). Several clinical reports (7, 33) have also indicated that the expression of adhesion molecules is increased in the duodenal mucosa of adult patients with food allergies or of infants with cow's milk-sensitive enteropathy. However, only a few studies have addressed lymphocyte trafficking in food allergies, and the mechanisms of directly "trafficking" cells have only scarcely been explored (11, 28). The 4/
7 heterodimer is the principal homing receptor that mediates the tissue-specific binding of lymphocytes to venules in the murine gut-associated lymphoid system by interacting with endothelial mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (3, 4, 15). It has also been shown that there is not simply one adhesion molecule with an exclusive function but rather that several have overlapping functions. L-selectin and CD11a/CD18, for example, play a role in the interaction between lymphocytes and high endothelial venules in the gut wall (1, 3). Therefore, whether lymphocyte trafficking is altered in food allergies, and if so which molecular interactions are responsible, should be determined.
The aim of this study was to establish an ovalbumin (OVA)-induced, chronic allergic model with histological changes in rats and to use the model to examine whether T cell homing is altered in the small intestine of rats with a food allergy. We monitored lymphocyte migration in Peyer's patches and in the small intestinal mucosa, using an intravital microscope, and our results indicated that vascular adhesion molecules, especially MAdCAM-1, participate in chronic T-cell migration to the intestine of rats with an OVA-induced allergy.
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MATERIALS AND METHODS |
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Assay of anti-OVA antibody and rat mast cell protease II concentrations. Blood samples collected from the abdominal aorta at death were coagulated at room temperature, then centrifuged for 20 min at 2,000 g at 4°C to obtain sera. Serum IgG and IgE specific for OVA were determined by ELISA as described by Knippels et al. (21). To detect OVA-specific-IgG, 96-well microtiter plates (Becton Dickinson) coated with OVA were incubated with PBS containing BSA and Tween 20, washed, and then incubated for 1 h at 37°C with serially diluted rat serum in PBS/BSA-Tween 20. Peroxidase-conjugated goat anti-rat IgG (hematoxylin and eosin) (KPL, Guildford, UK) in PBS/BSA-Tween 20 was added to the wells. After samples were washed, enzyme substrate 3,3',5,5'-tetramethylbenzidine (Sigma, St. Louis, MO) was added followed by H2SO4. Optical density was read spectrophotometrically at 450 nm using an ELISA plate reader (Benchmark Microplate Reader; Bio-Rad, Hercules, CA). To detect OVA-specific IgE, 96-well microtiter plates were coated with mouse anti-rat IgE (KPL). Rat serum samples were added to the plates followed by OVA-digoxigenin (DIG) conjugate (Roche Diagnostics). The plates were washed, and then peroxidase-conjugated sheep anti-DIG Fab fragment (Roche Diagnostics) in PBS/BSA-Tween 20 was added. Color was developed and measured as described for the OVA-specific IgG ELISA. Pooled sera from naive animals constituted the negative control (no OVA exposure). The reciprocal of the furthest serum dilution giving an extinction coefficient above the reference value was read as the titer. Serum concentrations of rat mast cell protease II (RMCPII) were assayed by using an enzyme-linked immunosorbent assay kit (Moredun Animal Health, Edinburgh, Scotland).
Histological evaluation and immunohistochemistry of the intestinal mucosa. Specimens for light microscopy were obtained from a location 10 cm toward the anal side from the pylorus (jejunum) and from a location 10 cm toward the oral side from the ileocecal valve (ileum). Samples were fixed with 10% formalin and stained with hematoxylin and eosin to evaluate morphological changes. Eosinophils were identified after Giemsa staining.
Another part of the intestine was fixed in periodate-lysine-paraformaldehyde for an immunohistochemical study by the labeled streptavidin biotin method. Intestinal tissues were vertically embedded in optimum cutting temperature compound (Sakura Fineteck, Tokyo, Japan) before being frozen in dry ice and acetone. Cryostat sections (7 µm thick) were transferred to poly-L-lysine-coated slides and air-dried for 1 h at 20°C. After being washed for 5 min in PBS (pH 7.4) containing 1% Triton X, sections were incubated in 5% normal goat serum in PBS. Primary antibodies against CD4 (OX35), CD8 (OX8), B cell (OX12), IL-2 receptor (OX39), MAdCAM-1 (OST-2), VCAM-1 (MR106), and ICAM-1 (1A29 [PDB] ) were obtained from BD PharMingen (San Diego, CA), and antibody against RMCPII was obtained from Biochem (London, UK). Sections were incubated with the second antibody, biotinylated conjugated anti-rat IgG, for 1 h at room temperature, followed by FITC-conjugated streptavidin (Amersham, London, UK) for 30 min at room temperature. After each of these steps, the sections were rinsed with PBS containing 1% BSA. A coverslip was applied by using glycerol jelly, and the sections were observed under a fluorescence microscope (model BX60; Olympus, Tokyo, Japan). Vessels positive for adhesion molecules in the lamina propria were calculated by using an image analyzer and quantified as the area of positively stained vessels per millimeter of muscularis mucosa. The infiltrated cells are expressed as the number of CD4, CD8, B cells, IL-2 positive cells, or RMCPII-positive cells per millimeter of muscularis mucosa.
In another set of experiments, monoclonal blocking antibody against MAdCAM-1 (OST-2; 2 mg/kg) or nonblocking antibody to MAdCAM-1 (OST-20; 2 mg/kg) (17) in 0.2 ml saline was administered intraperitoneally twice a week during the OVA challenge period (6 wk) to the daily gavage group, and histological changes in the intestine were compared.
Collection and labeling of T lymphocytes from intestinal lymph. The rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). The main mesenteric lymphatic duct was cannulated as described by Bollman et al. (5). Animals were then maintained in Bollman cages, and isotonic saline was infused intravenously via the jugular vein at 2.4 ml/h to replenish fluids and electrolytes lost due to lymphatic drainage. Lymph samples were collected in ice-cold vials containing heparin (6 U/ml), FBS, and RPMI 1640 medium (pH 7.4; GIBCO, Grand Island, NY). Lymphocytes from the mesenteric lymph were washed three times with working medium [RPMI 1640 medium (pH 7.4) containing penicillin and streptomycin (GIBCO) and 0.1% BSA] before separation and labeling. The T cell-rich fraction of lymphocytes from mesenteric lymphatics was obtained by using a nylon wool column. The whole cell population of 1 x 108 lymphocytes in 20 ml of RPMI 1640 medium containing 1% FBS was incubated at 37°C for 1 h with 1 g of nylon wool (Kanto Kagaku, Tokyo, Japan) in a column, and the pass-through fraction was designated as the T cell-rich fraction. These manipulations did not significantly affect lymphocyte viability as assessed by trypan blue exclusion. The cells were washed and resuspended in RPMI 1640 medium containing 5% FCS on ice. Lymphocytes were labeled with 15.6 mM carboxyfluorescein diacetate succinimidyl ester (CFDASE) (Molecular Probes, Eugene, OR) in DMSO. Immediately before infusion to recipient rats, lymphocytes (1 x 108) in 20 ml RPMI 1640 medium were incubated with 20 µl CFDASE for 30 min at 37°C.
Intravital observation of T lymphocyte migration at Peyer's patches. After an intraperitoneal injection of pentobarbital sodium (50 mg/kg), the abdomen was opened via a midline incision. Twelve centimeters of the ileal segment near the ileocecal valve was placed on a plastic plate for observation. The intestine was kept warm and moist by continuous superfusion with physiological saline warmed to 37°C. Two small incisions were made in the intestine, and warm Krebs-Ringer solution (pH 7.4) was instilled through a vinyl tube from the proximal end to maintain the luminal pressure at 15 cmH2O. Suitable areas of the microcirculation in Peyer's patches were observed from the serosal side using an intravital microscope (model Diaphot TMD-2S; Nikon, Tokyo, Japan) equipped with a silicon-intensified target image tube camera with a contrast-enhancing unit (model C-2400-08; Hamamatsu Photonics, Shizuoka, Japan) (25). Lymphocytes (6 x 107 cells in 1 ml RPMI 1640 medium) were injected into the jugular vein of the recipient rats during 3 min. The migration of CFSE-labeled T lymphocytes through the microvasculature of Peyer's patches was continuously recorded on S-VHS videotapes for up to 60 min after the injection. Epifluorescence was achieved by excitation at 470490 nm and emission at 520 nm.
The lymphocyte behavior in postcapillary venules (PCV) was classified according to the nature of interaction with the vascular walls. Lymphocytes adhering to venular walls but with movement along them were defined as "rolling." The ratio of rolling lymphocytes per total cell flux was calculated. Lymphocytes adhering to the venular wall without movement for >30 s were defined as "sticking" lymphocytes. The number of sticking lymphocytes was determined in 1-mm2 observation fields that contained PCV of 2550 µm in diameter.
In some experiments, anti-MAdCAM-1 MAb (OST-2; 2 mg/kg) or anti-ICAM-1 (1A29 [PDB] ; 2 mg/kg) in 0.2 ml saline was infused from the jugular vein 30 min before starting the infusion of T lymphocytes. The control was a nonblocking antibody to MAdCAM-1 (OST-20) or an isotype-matched IgG applied under the same conditions (17).
Intravital observation at villus mucosa. We examined the microcirculation of intestinal villi from the mucosal surface and observed lymphocyte migration in another set of experiments. A segment of the ileum was gently extended onto a plate, and a longitudinal incision was cut along its antimesenteric border by microcautery. The behavior of fluorescence-labeled lymphocytes in villus tips was visualized on a video monitor through a fluorescence microscope as described (26). The tip of each villus was observed as an oblique circle, and villus microvessels were observed. As in the observations of Peyer's patches, lymphocytes that remained in the same position for over 30 s were defined as sticking.
Statistics. All results are expressed as means ± SE. Differences among groups were evaluated by ANOVA and Fisher's post hoc test. Statistical significance was established at P < 0.05.
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RESULTS |
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Histological evaluation and immunohistochemical study of intestinal RMCPII, lymphocyte subpopulation, and adhesion molecules. Table 1 shows jejunal and ileal morphology determined by light microscopy. The villus height of the daily gavage group was significantly reduced and crypt depth was increased with inflammatory cell infiltrations compared with the OVA-sensitized and OVA-fed controls at both sites (Fig. 2A). This was also true of the free-drinking group, although these changes were less significant compared with the daily gavage group. The numbers of eosinophils were increased in the mucosa of both regions of the intestine of rats exposed to OVA compared with OVA-sensitized and OVA-fed controls.
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The serum concentrations of RMCPII were significantly increased in rats exposed to OVA compared with controls. The RMCPII concentrations in the daily gavage and free-drinking groups were 2.3- and 1.1-fold greater, respectively, compared with OVA-sensitized controls (P < 0.05). Figure 3 shows counts of mucosal mast cells determined by using anti-RMCPII antibody in the small intestinal mucosa. The numbers of mucosal mast cells were significantly increased in the mucosa of both intestinal regions of daily gavage groups compared with OVA-sensitized or OVA-fed controls. However, mast cell numbers did not significantly differ between free-drinking and control groups.
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We determined the effect of chronic OVA exposure on lymphocyte subpopulations in the intestinal mucosa by immunohistochemical means (Table 2). Figure 2B shows that the CD4 lymphocyte population was significantly increased in the daily gavage group exposed to OVA compared with either OVA-sensitized or OVA-fed controls both in the proximal and the distal small intestinal mucosa. The numbers of CD4 cells were also significantly increased in the free-drinking group, although to a lesser extent than in the daily gavage group, especially in the ileum. However, the numbers of CD8 lymphocytes were significantly decreased in both groups exposed to OVA compared with either of the controls at both sites. The numbers of OX12-positive B cells were significantly increased in the groups exposed to OVA compared with either of the controls. On the other hand, the numbers of IL-2 receptor-positive T cells did not significantly differ between control and daily gavage groups.
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To determine whether the expression of adhesion molecules in intestinal microvessels is enhanced to induce lymphocyte migration, we stained jejunal and ileal mucosa with antivascular adhesion molecules using an immunohistochemical technique. Figure 2C shows that MAdCAM-1 was mainly expressed in the endothelium of venules in the lamina propria of the intestinal mucosa just above the muscularis mucosa. However, daily gavage exposure to OVA induced a significant increase in MAdCAM-1 expression. This adhesion molecule was mainly induced in the lamina propria at the base of crypts, but its expression was also increased in some microvessels of the upper villus and in the submucosa. Table 3 shows a quantitative analysis of three adhesion molecules determined as the area of positively stained vessels per millimeter of muscularis mucosa in the small intestinal mucosa. MAd-CAM-1 expression was significantly induced in both the proximal and the distal parts of the intestine of free-drinking or daily gavage groups compared with OVA-sensitized controls. On the other hand, the expression of ICAM-1 or VCAM-1 was less notable in the small intestine and did not significantly change after chronic OVA exposure.
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We examined whether antibodies against MAdCAM-1 could affect the histological changes in the small intestinal mucosa of chronically allergic rats. Figure 4A shows that the functional blocking of MAdCAM-1 significantly attenuated the reduced villus height and the increased crypt depth induced by chronic OVA exposure compared with the control antibody groups at both sites. Figure 4B shows that anti-MAdCAM-1 blocking antibody, but not nonblocking antibody, significantly inhibited the increased numbers of CD4 cells in the intestinal mucosa of rats chronically exposed to OVA. However, the numbers of CD8 cells did not significantly differ between these two groups.
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Lymphocyte migration to PCVs of Peyer's patches and villus mucosa. We examined whether T lymphocyte migration to the small intestinal mucosa is induced in chronic allergic animals exposed to OVA. Because cell-mediated immunity was more obvious in the daily gavage groups, we compared the effects in these animals with those in OVA-sensitized controls. We examined the in vivo migration of T lymphocytes to both lymphoid (Peyer's patches) and nonlymphoid (intestinal villus mucosa) areas of the small intestine. After an infusion of fluorescence-labeled T lymphocytes, the total numbers of lymphocytes that entered PCVs within 10 min of infusion into the jugular vein did not significantly differ between control and gavaged animals (58.5 ± 15.7 vs. 58.3 ± 13.3 per minute). Some lymphocytes showed characteristic rolling behavior on the surface of the endothelial wall in the PCVs of Peyer's patches, and the ratio (%) of rolling cells in daily gavage groups (61.8 ± 8.6) was much greater than that in controls (37.1 ± 8.9) 10 min after infusion. These findings indicated that lymphocyte-endothelial interaction was greater in the allergic rats. Some lymphocytes adhered to the endothelial walls of PCVs. The representative photomicrographs in Fig. 5A show that the number of T lymphocytes sticking in Peyer's patches 20 min after infusion was remarkably increased in allergic rats and reached about double and 1.7-fold the control numbers at 20 and 40 min, respectively (Fig. 6A).
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We also visualized interactions between CFSE-labeled T lymphocytes and microvessels in the small intestinal mucosa. The total number of lymphocytes that entered intestinal microvessels did not significantly differ between control and chronic allergic rats at 10 min after infusion (35.3 ± 9.6 vs. 33.3 ± 7.6/min). Figure 5B shows microscopic images of the distribution of T lymphocytes sticking in the intestinal (ileal) mucosa 30 min after infusion. A few T lymphocytes adhered to the intestinal microvessels of control rats, whereas in the daily gavage groups, adherence was significantly increased and reached 3.7- and 2.7-fold the control numbers at 20 and 40 min, respectively (Fig. 6B).
We examined the effect of antibodies against adhesion molecules on T lymphocyte migration in the PCV of Peyer's patches and in the small intestinal mucosa of chronic allergic rats. Figure 6 shows that functionally blocking adhesion molecules inhibited the numbers of T lymphocytes that adhered to intestinal microvessels (PCV of Peyer's patches and villus microvessels) at 20 and 40 min. The number of T lymphocytes was significantly decreased by a prior infusion of anti-MAd-CAM-1 to rats in both the PCV of Peyer's patches and the small intestinal mucosa. Nonblocking antibody to MAdCAM-1 (OST-20) did not affect the T lymphocyte sticking (data not shown). Nevertheless, the significant inhibitory effect of anti-MAdCAM-1 antibody on adherence of T lymphocytes to the PCV of Peyer's patches and villus microvessels (85.3 ± 6.0 and 89.2 ± 8.0% inhibition, respectively) was also observed in naive animals, suggesting that the effects are not restricted to the inflammatory state induced by feeding antigen. On the other hand, anti-ICAM-1 antibody did not significantly attenuate T lymphocyte interactions stimulated by exposure to OVA in either area.
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DISCUSSION |
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Humans frequently develop allergic reactions to dietary proteins. The BN rat is a high immunoglobulin responder strain with a genetic predisposition to overproduce IgE in response to antigens. We reasoned that hyperstimulation of the IgE response would disturb the mucosal immune mechanisms to result in a cell-mediated reaction against exposure to oral antigens. Our model free-drinking and daily gavage groups both developed IgE-dependent intestinal allergies, because a specific IgE response was identified and RMCPII levels were increased in the serum of these animals. However, cell-mediated immunity was more obvious in the daily gavage group along with specific IgG, suggesting a Th1-like response. In our preliminary study of delayed-type hypersensitivity (DTH) determined by using the ear-swelling test, a significant DTH response developed in the daily gavage group and to a lesser extent in the free-drinking group (data not shown). These results are in accordance with significant lymphocyte infiltration of the intestinal mucosa in response to allergen challenge.
We speculate that if cell-mediated reactions are induced by the chronic phase of the IgE-mediated allergic response, then a T-cell-mediated process may largely contribute to the pathogenesis of mucosal morphological changes. Cell-mediated reactions on the intestinal mucosa have been documented in detail during the graft vs. host reaction (23). This hypothesis is supported by the finding in the mouse model of food-sensitive enteropathy, in which migrated lymphocytes in the intestinal mucosa can proliferate in response to stimulation with a specific antigen (29). The increased number of lymphocytes in the intestinal mucosa is thought to be the result of lymphocyte recruitment from the blood into the intestine. To investigate this possibility, we performed the in vivo migration study on T lymphocytes and uncovered direct evidence that T lymphocyte migration to both the intestinal Peyer's patches and villus mucosa was stimulated after chronic antigen exposure. We also demonstrated that these increases in T lymphocyte migration to small intestinal mucosa might be brought about via MAd-CAM-1 on the microvascular endothelium, because prior exposure to this adhesion molecule significantly attenuated T-lymphocyte migration in both the PCV of Peyer's patches and in villus microvessels. The significant increase in MAdCAM-1 expression induced by chronic OVA exposure supports this notion. On the other hand, we did not find evidence that either ICAM-1 or VCAM-1 is involved in T lymphocyte migration in the small intestine. Several studies (7, 11, 33) on food allergy and animal models have suggested that the increased expression of ICAM-1 or VCAM-1 is related to the pathogenesis of leukocyte infiltration to the intestinal mucosa. We surmise that leukocyte subpopulations other than T cells, such as neutrophils, might play important roles via an ICAM-1-dependent mechanism in these situations.
Activated mast cells that produce sustained IgE responses might also contribute to the mechanism of enhanced lymphocyte migration. TNF- produced from mast cells might be an important factor for the upregulation of adhesion molecules including MAdCAM-1 (16, 35). To our knowledge, this is the first direct demonstration of lymphocyte migration to gut-associated lymphoid organs and intestinal mucosal microvessels in situ in a chronic food allergy model. However, the exact mechanism, including interactions between chemokines and chemokine receptors with integrins, remains to be further investigated. The increased trafficking of lymphocytes to the intestine exposed to antigen together with abnormal cell-mediated immune responses and IgE-mediated mast cell stimulation in situ might play a key role in the development of intestinal mucosal injury in patients with chronic food allergies.
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ACKNOWLEDGMENTS |
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This study was supported, in part, by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture of Japan and by grants from Keio University School of Medicine and the National Defense Medical College.
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FOOTNOTES |
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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.
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REFERENCES |
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