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Keywords:

  • Crohn's disease;
  • IL-5;
  • Eosinophil;
  • Th2;
  • IBD

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

SAMP1/Yit mice spontaneously develop ileitis resembling Crohn's disease (CD) without chemical or genetic manipulations. Since the focus of studies were Th1 cytokines, only Th1-type T cells were thought to be responsible for intestinal inflammation in these mice. To further characterize the pathogenesis of this ileitis, we investigated the implication of Th2 cytokines in ileitis of SAMP1/Yit mice. The expression of chemokine receptors (CCR) associated with both Th1 and Th2 lymphocytes, such as CCR2, CCR3, CCR4, CCR5, and CCR8, was increased. Among cytokines, IL-5 was remarkablyincreased in Peyer's patches, mesenteric lymph nodes, and mucosa involved in ileitis. Furthermore, infiltration of numerous eosinophils in ileitis was histologically evident. Severe combined immunodeficiency mice injected intraperitoneally with CD4+ cells from SAMP1/Yit mice developed colitis and ileitis, with the infiltration of eosinophils. Administration of anti-IL-5 antibodies significantly attenuated ileitis in these mice. We suggest that IL-5 participates in the pathogenesis of ileitis and that anti-IL-5 antibodies are potentially useful for immunotherapy in CD patients. This is the first demonstration that IL-5 is crucial for the development of ileitis in this mouse model of CD.

Abbreviations:
CD:

Crohn's disease

CCR:

Chemokine receptor

H&E:

Hematoxylin and eosin

hpf:

High-power field

IBD:

Inflammatory bowel disease

MLN:

Mesenteric lymph nodes

MN:

Mononuclear cell

PP:

Peyer's patches

RT:

Reverse transcription

SCID:

Severe combined immunodeficiency

UC:

Ulcerative colitis

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Crohn's disease (CD) and ulcerative colitis (UC) constitute two major categories in human inflammatory bowel disease (IBD). Cytokine balance in the mucosa is likely to be important in thedevelopment of IBD 1, 2. Various experimental models mimic important aspects of human IBD 3, 4. Colitis in IL-10 knockout (KO) mice resulted in granuloma formation 5, a characteristic feature of CD; such colitis occurs via a Th1 pathway 6. On the other hand, production of Th2 cytokines, mainly IL-4, was increased in UC-like models, such as T cell receptor α chain KO mice and mice given oxazolone 7, 8. IL-4 was also shown to be important inthe induction of crypt inflammation resembling UC in trinitrobenzene sulfonic acid (TNBS)-induced colitis 9.

Recent clinical studies have shown different cytokine profiles in CD and UC 10. It is generally recognized that Th1 cytokines are mainly involved in CD, whereas Th2 cytokines may have a more significant role in UC. In human CD, activated eosinophils and increased amounts of IL-5 (a Th2 cytokine) were also reported 11, 12. IL-5 is themain mediator for eosinophil recruitment and activation 13. IL-5 and eosinophils are involved not only in allergic diseases (such as atopic dermatitis, asthma, and rhinitis) but also in inflammatory diseases (such as IBD and eosinophilic gastroenteritis) 14. However, the specific roles of IL-5 and eosinophils in the pathogenesis of IBD have not been elucidated.

The SAMP1/Yit mouse, a unique model of intestinal inflammation, spontaneously develops a chronic ileitis resembling human CD 15. This ileitis shows discontinuous but transmural inflammation involving mainly the terminal ileum, and granuloma formation is prominent. Recently, several reports showed similarities between associated immune responses in these mice and in human CD. Anti-tumor necrosis factor (TNF)-α antibody and antibiotics therapy ameliorated intestinal inflammation in SAMP1/Yit mice as well as in human CD 1618.Adoptive transfer experiments showed that mesenteric lymph node (MLN) cells harvested from SAMP1/Yit mice were capable of transferring ileitis to severe combined immune deficiency (SCID) recipients in a dose- and time-dependent manner 16. SCID mice injected with CD4+ T cells also developed severe colitis. The fact that down-regulation of Th1 cytokine production and reduction of CD4/CD45RB T cells in MLN were observed to occur in response to these therapies supports the possibility that pathogenic CD4 T cells having a Th1-type profile could mediate ileitis in SAMP1/Yit mice. However, most studies have been conducted on Th1 cytokines, and the implication of Th2 cytokines, especially IL-5, in this mouse model of human CD has not been determined.

In the present experiments, to determine whether ileitis in SAMP1/Yit mice has similar features as human CD, we further investigated the cytokine balance in this ileitis. We have shown here the implication of Th2 cytokines, in particular IL-5, as well as Th1 cytokines in ileitis in SAMP1/Yit mice. We have also demonstrated for the first time that anti-IL-5 therapy in a mouse model of human CD significantly reduced the severity of ileitis by the inhibition of intestinal Th1 and Th2 cytokine production.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Cytokine and chemokine expression in the terminal ileum of SAMP1/Yit mice

Ileitis was developed in SAMP1/Yit mice at 10 weeks of age 15, and incidence and severity worsened with increasing age. SAMP1/Yit mice were killed at 8, 12, or 24 weeks of age and AKR/J mice at 24 weeks of age, and the terminal ileum was removed. Ileitis was not detectable in either AKR/J mice or SAMP1/Yit mice at 8 weeks. Ileitis was very mild at 12 weeks and severe at 24 weeks. To investigate the Th1/Th2 balance in the mucosa of the terminal ileum, we examined the cytokine mRNA expression by reverse transcription (RT)-PCR. mRNA expression of interferon (IFN)-γ, TNF-α, and IL-5 was increased at 24 weeks of age in SAMP1/Yit mice, and these increases were associated with the severity of ileitis (Fig. 1A). Furthermore, chemokine receptor (CCR) mRNA expression was examined in this model with respect to Th1 vs. Th2 cells. Previous studies have shown that CCR5 is involved in regulation of Th1 lymphocyte function and is expressed exclusively on Th1 cells 19, and that CCR2 is directly involved in induction of IFN-γ expression by T cells 20. On the other hand, CCR3, CCR4, and CCR8 are expressed by Th2 cells 21, 22. CCR3 is expressed by eosinophils as well as Th2 cells 23. We demonstrated that CCR2 and CCR5 mRNA as Th1-type chemokine receptors were increased in severe ileitis, and CCR3, CCR4, and CCR8 mRNA as Th2-type chemokine receptors were also increased in mice that developed severe ileitis (Fig. 1B). Therefore, not only Th1 cells but also Th2 cells were involved in ileitis.

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Figure 1. Analysis of cytokines and chemokine receptors in the terminal ileum. RNA from the mucosa of the ileum was used for RT-PCR. PCR cycling conditions were 36 s at 96°C, 1 min at 60°C, and 1 min at 72°C for 35 cycles after 5 min at 94°C. At the end of 35 cycles, samples were held at 72°C for 10 min. (A) mRNA levels of β-actin, IFN-γ, TNF-α, and IL-5 were determined in AKR/J mice at 24 weeks of age and SAMP1/Yit mice at 8, 12, and 24 weeks of age. (B) mRNA levels of CCR2, CCR3, CCR4, CCR5, and CCR8 were also determined.

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2.2 Eosinophil infiltration in ileitis in SAMP1/Yit mice

Since IL-5 has been reported to induce eosinophils in tissues 23, we examined histologically whether eosinophils were involved in ileitis. The Peyer's patches (PP) of AKR/J and SAMP1/Yit mice were similar at 24 weeks of age (data not shown). The ileum of AKR/J mice appeared normal (Fig. 2A, B), whereas SAMP1/Yit mice showed ileitis with infiltration not only by mononuclear cells (MN) but also by numerous eosinophils (Fig. 2C–F). These eosinophils in mucosa involved in ileitis were present throughout the length of the villi; in the normal state, eosinophils were limited to the base of the villi in the region of the crypt 24. When eosinophils were counted in the ileum at 24 weeks (Fig. 3A), an ileum without inflammation in AKR/J and SAMP1/Yit mice showed 0.61±0.2 eosinophils/villus and 0.5±0.2 eosinophils/villus, respectively (Fig. 3B). On the other hand, eosinophils were significantly increased in the inflamed ileum in SAMP1/Yit mice (8.2±1 eosinophils/villus) (Fig. 3C). There were very few eosinophils in the peripheral blood of AKR/J and SAMP1/Yit mice with or without ileitis (data not shown).

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Figure 2. Histological findings (H&E staining) at 24 weeks of age. (A, B) The terminal ileum of an AKR/J mouse shows normal mucosal features. (C–F) The terminal ileum of an SAMP1/Yit mouse shows severe ileitis with infiltration by PMN (including eosinophils) and MN. (Original magnification: A, C, ×100; B, D, ×200; E, ×400; F, ×1000).

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Figure 3. The number of eosinophils in the ileum. (A) Eosinophils in the ileum were counted from the base to the tip of 15 villi cut in a longitudinal section. The results are expressed as the number of eosinophils per villus (open bar, AKR/J mice at 24 weeks of age; dotted bar, SAMP1/Yit mice at 12 weeks; solid bar, SAMP1/Yit mice at 24 weeks). Data are expressed as means ± SEM (n=8). (B) Histology of the ileum in AKR/J mice at 24 weeks. (C). Histology of the ileum in SAMP1/Yit mice at 24 weeks. Eosinophils (arrow) were found in a villus. (Original magnification: B, C, ×400).

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2.3 IL-5 in inflamed ileum, MLN, and PP

IL-5 and CCR3 were investigated in various intestinal locations, such as duodenum, jejunum, ileum without inflammation, ileum with inflammation, cecum, and colon in SAMP1/Yit mice at 24 weeks of age. IL-5 and CCR3 mRNA were remarkably increased in the inflamed ileum (Fig. 4A). Thus, the increased number of eosinophils together with the increase in IL-5 represented a specific reaction at the inflammatory site of SAMP1/Yit mice. Since we hypothesized that this inflammation in the ileum of SAMP1/Yit mice might be the result of dysregulation of the mucosal immune system, we investigated associations between ileitis and characteristics of the PP and MLN. We found that the PP and MLN were markedly hyperplastic in mice with ileitis, and the number of cells obtained from the nodes increased with the development of ileitis. The number of PP cells from AKR/J age-matched control mice and SAMP1/Yit mice without or with ileum inflammation was 851±85, 825±66, and 1166±186, respectively (Fig. 4B); MLN cells numbered 1216±132, 1316±81, and 2150±216, respectively. A large percentage of the increased number of cells were CD4+ T cells, and CD4+ cells harvested from MLN of SAMP1/Yit mice at 24 weeks expressed a profile of surface markers consistent with an activated phenotype (Table 1), although the expression levels of CD25 and CD62L were not significant. Representative results are shown in Fig. 4C. In contrast, CD4 cells from MLN of SAMP1/Yit mice at 8 weeks expressed a naïve phenotype. When compared with AKR/ J mice at 24 weeks, the expression profile of CD4+ T cells from age-matched control SAMP1/Yit mice was not significantly activated (Fig. 4C). In addition, the increase of IL-5, IFN-γ, and TNF-α mRNA in the PP and MLN was clearly associated with the severity of ileitis, although CCR3 mRNA showed no difference (Fig. 4D). We examined the production of IL-5, IFN-γ, and TNF-α by PP and MLN after stimulation with anti-CD3 (Fig. 5) and found that IL-5 production was increased in SAMP1/Yit mice at 24 weeks of age at the protein level as well as at the mRNA level. Production of IFN-γ and TNF-α at the protein level was also increased, although the extent of increase of these cytokines was lower than that of IL-5. Taken together, these data imply that IL-5-secreting T cells from the PP and MLN are involved in the pathogenesis of ileitis, as well as IFN-γ- or TNF-α -secreting T cells.

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Figure 4. Analysis of IL-5 and CCR3 expression in the intestine, PP, and MLN. (A) IL-5 and CCR3 expression in a SAMP1/Yit mouse at 24 weeks of age. RNA from the mucosa of each intestine was used for RT-PCR. Inflammation was present mainly in the terminal ileum, with other regions showing no inflammation or very mild inflammation. (B) Cell numbers in PP and MLN (n=8). Cell number in SAMP1/Yit mice at 24 weeks of age (solid bar) was greater than in AKR/J mice at 24 weeks (open bar) or SAMP1/Yit mice at 12 weeks (dotted bar). (C) Analysis of surface marker expression by CD4+ T cells from MLN. Cells of MLN were harvested from 8- or 24-week-old SAMP1/Yit or AKR/J mice and analyzed for the expression of CD25, CD69, CD62L, and CD45RB in CD3+- and CD4+-gated cells (n=5). (D) RNA from freshly isolated MLN and PP cells were used for RT-PCR. mRNA levels of IL-5, IFN-γ, TNF-α, and CCR3 were determined in AKR/J mice at 24 weeks of age and in SAMP1/Yit mice at 12 and 24 weeks.

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Table 1. Phenotyping of CD4+ T cells from MLNa)
Phenotype8W SAMP1/Yit24W SAMP1/Yitp
  1. a) The mean fluorescence intensity (MFI) of surface markers was evaluated.

  2. b) MFI ± SEM, n=5. NS, not significant.

CD45RB29.6±2.2b)18.8±2.0<0.05
CD25 3.9±0.7 9.0±1.3NS
CD62L10.3±0.917.4±2.8NS
CD69 4.4±0.623.2±2.8<0.05
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Figure 5. Cytokine production of IL-5, IFN-γ, and TNF-α in PP and MLN cells. PP or MLN cells were cultured for 72 h in the presence or absence of an anti-CD3 mAb. The concentration of IL-5, IFN-γ, and TNF-α in the culture supernatants was examined by specific ELISA. IL-5, IFN-γ, and TNF-α production by PP and MLN was increased by stimulation with the anti-CD3 mAb (p<0.01). Data are expressed as means ± SEM (n=8).

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2.4 Anti-IL-5 antibody attenuated ileitis in a cell transfer model

Adoptive transfer experiments showed that MLN cells harvested from SAMP1/Yit mice were capable of transferring ileitis to SCID recipients 16. In adoptive transfer mice receiving MLN cells of SAMP1/Yit mice, MN and some PMN, mainly eosinophils, were present throughout the length of the small intestine (Fig. 6A–D) and the colon. In contrast, adoptive transfer mice receiving MLN cells of AKR/J mice showed no infiltration of inflammatory cells throughout the small intestine (Fig. 6E). Although several reports implicated pathogenic Th1-type cells in ileitis, pathogenic T cells from SAMP1/Yit mice presumably produced IL-5, and this Th2 cytokine appeared to be important in the pathogenesis of ileitis in this model as well. To examine this hypothesis, we injected SCID mice twice with an anti-IL-5 antibody, 2 h before injecting CD4+ T cells and again 3 weeks afterwards. A recent report showed that administration of 30 μg of anti-IL-5 antibody prevented infiltration of eosinophils in bronchoalveolar lavage fluid in a murine asthma model 25. In our animals, anti-IL-5 antibody administration significantly reduced the infiltration of MN and polynuclear cells (Fig. 6F), prevented weight loss (Fig. 7A), and decreased the inflammatory index (Fig. 7B) compared with mice treated with isotypic control antibody. Furthermore, IL-5, IFN-γ, and TNF-α expression was significantly reduced in mice treated with the anti-IL-5 antibody (Fig. 7C).

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Figure 6. Histology of the ileum in SCID mice injected with CD4+ T cells in addition to intraperitoneal administration of an anti-IL-5 mAb or an isotype control antibody. Histological features of ileitis adoptively transferred to SCID recipients in combination with the isotype control antibody showed severe inflammation with the infiltration of eosinophils and MN (A–D). SCID recipients of CD4+ T cells from AKR/J mice showed no inflammation (E). In the ileum of adoptively transferred SCID mice treated with the anti-IL-5 mAb, inflammation was ameliorated (F). (Original magnification: A, ×100; B, ×200; C, ×400; D, ×1000; E, ×200; F, ×200).

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Figure 7. Attenuation of inflammation in the ileum and colon in SCID mice injected with CD4+ T cells by intraperitoneal administration of anti-IL-5 mAb. (A) Body weight of SCID mice injected with CD4+ T cells from SAMP1/Yit or AKR/J mice. The weight loss of SCID mice that were transferred with CD4+ T cells from SAMP1/Yit mice was significantly decreased by treatment with the anti-IL-5 mAb compared to that after treatment with control IgG (p<0.05 by ANOVA with Turkey's test). n=12. The arrow indicates the timing of the treatment. (B) Inflammatory index assessed by summing scores for acute and chronic inflammation. Untreated SCID mice had severe inflammation with both PMN, mainly eosinophils, and MN in the ileum and colon. In contrast, SCID mice treated with anti-IL-5 showed mild inflammation (p<0.05). Data are expressed as means ± SEM (n=12). (C) IL-5, IFN-γ, and TNF-α mRNA expression in the mucosa of the ileum from cell-transferred SCID mice treated with an anti-IL-5 mAb or the isotype control antibody was measured by RT-PCR.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

It is generally recognized that Th1 cytokines are mainly involved in CD, whereas Th2 cytokines may have a more significant role in UC. In most murine models resembling human CD, as well as in patients with CD, IL-12 activates Th1 cells in inflammatory lesions where they drive local inflammation by producing IFN-γ and TNF-α 3. Recent studies showed that human CD patients and SAMP1/Yit mice shared similar pathogenetic mechanisms of disease, and Th1 type cells have been considered the center of the pathogenesis of this ileitis 15, 16. Amelioration of ileitis by anti-TNF-α antibodies was reported in SAMP1/ Yit mice as a consequence of inhibiting Th1 cytokines 16, 17, 26. In our preliminary experiment, we confirmed these results (data not shown). Most of the studies have focused on Th1 cytokines, and the role of Th2 cytokines, especially IL-5, in this mouse model of human CD has not been determined. In the present experiments, we implicated both Th1 and Th2 cells in the ileitis of SAMP1/Yit mice, demonstrating increases not only in Th1 cytokines but also in Th2 cytokines, mainly IL-5.

As for the ileitis in human CD, foods are known to be one factor exacerbating ileitis. It is possible that an allergic reaction to antigens such as foods or bacteria would be mediated by eosinophils in the inflamed human ileum, since IL-5 and eosinophils are often involved in allergic disease. Indeed, increased amounts of IL-5 and eosinophil infiltration in human CD were reported 11. Activated eosinophils release specific cationic proteins, cytotoxic for various targets including intestinal epithelial cells. Since eosinophil activation and the presence of IL-5 mRNA were seen in early endoscopically detected recurrence of CD 11, it has been thought that local synthesis of IL-5 associated with eosinophil activation in the tissues could participate in early mucosal damage in CD 11. However, the mechanisms that trigger eosinophil activation and cytokine synthesis have not been elucidated. In our study, we histologically detected that numerous eosinophils infiltrated the mucosa in case of ileitis. Importantly, SCID mice injected with CD4+ T cells from the MLN of SAMP1/Yit mice developed ileitis and colitis characterized by infiltration with numerous eosinophils. Neutralization of IL-5 resulted in ameliorated eosinophil infiltration associated with ileitis. Thus, IL-5 secreted by CD4+ T cells could have recruited and activated eosinophils and induced inflammation in the mucosa.

Interaction between epithelial and lymphoid elements in the PP is a characteristic feature of the mucosal immune system. Luminal antigens must gain access to the gut-associated lymphoid tissues (GALT) via M cells in the PP epithelium. In this study, IL-5 production and the number of cells in ileitis-affected PP, MLN, and mucosa was significantly associated with the severity of ileitis. CD4+ T cells from not only MLN but also PP of SAMP1/Yit mice caused severe ileitis. CD4+ T cells from PP also expressed activated phenotypes (data not shown). These results suggest that PP are important for inducing active immunity in the development of ileitis. Further studies would be needed to elucidate the roles of PP in the development of disease.

Our data suggested that IL-5, which is a Th2 cytokine, is important for inducing inflammation of the ileum by recruiting and stimulating PMN such as eosinophils. We hypothesize that in our SCID mice, transferred CD4 T+ cells from SAMP1/Yit mice migrate to the intestine and drive the local inflammation through production of IL-5. Administration of an anti-IL-5 antibody significantly attenuated ileitis in SCID mice and might have reduced the eosinophil influx in the intestine by inhibiting local effects of IL-5 in the development of ileitis. Still, the effect of the anti-IL-5 antibody was limited; since ileitis was not fully ameliorated, a combination therapy with other reagents would offer a therapeutic advantage in the treatment of ileitis. In future studies, we would like to determine whether or not the anti-IL-5 therapy could show an effect also on SAMP1/Yit mice. In this model, neutralization of IL-5 could lead to down-regulation of IFN-γ and TNF-α production, suggesting that IL-5 may also participate in Th1 recruitment.

With regard to our results when comparing the diseased mice to AKR/J mice, the expression profile of CD4+ T cells from MLN was not as significantly activated as previously reported byothers 16, 17. However, CD4+ T cells from diseased mice seemed to have a significantly activated phenotype when compared to that from SAMP1/Yit mice at 8 weeks. Only CD4+ T cells from SAMP1/Yit mice caused inflammation when MLN CD4+ T cells from either SAMP1/Yit and AKR/J mice were transferred to SCID mice.

The results of our study include the important finding that ileitis in SAMP1/Yit mice has features similar to those of human CD with regard to the participation of IL-5, eosinophils, and Th1 cytokines. This is the first demonstration that IL-5 participates in ileitis in this mouse model. Considering our results together with those of earlier studies, the SAMP1/Yit mouse model most closely resembles the human condition and offers the opportunity to study the effects and mechanisms of action. We demonstrated that an anti-IL-5 therapy in a mouse model of human CD significantly reducedthe severity of ileitis by the inhibition of both intestinal Th1 and Th2 cytokine production, although the mechanism of down-regulation of Th1 cytokines by neutralization of IL-5 has not yet been clarified. The results of this study could have important implications for the treatment of CD. Our findings suggest that an anti-IL-5 antibody therapy has the potential of a new treatment for CD patients.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Mice

SAMP1/Yit mice (Yakult Central Institute for Microbiological Research, Tokyo, Japan) were maintained under specific pathogen-free conditions at Kurume University. The SAMP1/Yit (H-2k)strain originated from AKR/J mice (Seiwa Experimental Animals Ltd., Fukuoka, Japan). SCID (C3HSmn.CB17-Prkdcscid/J) mice were purchased from The Jackson Laboratories (Bar Harbor, ME), to serve as cell transfer models. These studies were approved by the Institutional Animal Care Committee.

4.2 Histological assessment of ileitis

Mice were killed at 8, 12, and 24 weeks of age, and the terminal ileum was removed. For histological examination, specimens were fixed in 3% buffered formalin and embedded in paraffin. Multiple 4-μm sections were stained with hematoxylin and eosin (H&E) using standard techniques and microscopically scored in a semi-quantitative manner as described 16. For acute inflammation, scores were 0=0–1 PMN (mainly eosinophils) per high-power field (hpf) (×400) within the mucosa; 1=2–10 PMN/hpf; 2=11–20 PMN/hpf; 3=21–30 PMN/hpf; and 4=>30 PMN/hpf. For chronic inflammation, scores were 0=0–10 MN/hpf within the mucosa; 1=11–20 MN/hpf; 2=21–30 MN/hpf; 3=31–40 MN/hpf; and 4=>40 MN/hpf. Acute and chronic inflammatory scores were added to result in a total histological score. In the ileum, eosinophils were randomly counted from the base to the tip of 15 villi cut in a longitudinal section, and the result was expressed as the number of eosinophils per villus.

4.3 RT–PCR

Total RNA was extracted from the mucosal layer without the outer muscle layer of duodenum, jejunum, ileum, cecum, and colon 27. After cutting and washing these intestinal segments, the peritoneal aspects of the intestine were placed on a plastic plate and exposed to dry ice for 10 s to freeze the muscle layer but not the mucosa. Blades were used to separate the mucosaltissue. In addition to mucosa, total RNA was extracted from PP and MLN after gentle cell dispersion using 25-gauge needles and passage through a nylon membrane (60 μm). Following extraction of 2 μg of RNA from the mucosa and total RNA from 2×106 PP cells and 2×106 MLN cells by the acid guanidinium thiocyanatephenol-chloroform method, the RNA was converted to cDNA using the oligo(dT) primer in a 20-μl RT reaction mixture (GIBCO BRL, Gaithersburg, MD). Then, 1 μl of the RT reaction was amplified by use of a hot-start PCR protocol with specific primers. The primers used for determining cytokine expression were reported by others 28. The oligonucleotides for determination of CCR expression were: CCR2 (sense, 5′-CTA TCA ACA TCT CAT TCT CTA TTT A-3′; antisense, 5′-CCC AAA GAC CCA CTC ATT TGC AGC-3′); CCR3 (sense, 5′-CTG CTA CTC AGG AAT CAT TAA AAC-3′; antisense, 5′-GTT CTT TCC ATT TTC TCA CCA GGA AG-3′); CCR4 (sense, 5′-GGC AAG GAC CCT GAC CTA TGG GGT CAT CAC-3′; antisense, 5′-GTG CAG TCC TGA AGG GAC TTC AAG CTC CAC CAG-3′); CCR5 (sense, 5′-TACCAG ATC TCA GAA AGA AGG TTT TCA TTA-3′; antisense, 5′-GCG TTT GAC AAT GTG TTT TCG GAA GAA CAC T-3′); CCR8 (sense: 5′-GTG GTC TCT GGC CTT TAT TAC ATT GG-3′; antisense, 5′-ACA TCC ATC CAA GAT GTG CAG GTC-3′); β-actin (sense, 5′-GTG GGC CGC TCT AGG CAC CA-3′; antisense, 5′-CGG TTG GCC TTA GGG TTC AGG GGG G-3′).

4.4 Flow cytometry

Cells in suspension were labeled with fluorochrome-tagged antibodies including anti-CD3, -CD4, -CD8, -CD69, -CD25, -CD62L, and -CD45RB (PharMingen, San Diego, CA). Cells were analyzed after fixation with 1% formalin. To determine the percentage of cells expressing surface markers and the intensity of expression, three-color analyses were performed using a FACS system (Beckman Coulter, Fullerton, CA).

4.5 Cytokine assay

To measure cytokine production, 24-well plates (Corning, Corning, NY) were coated with 10 μg/ml hamster anti-murine CD3ϵ antibody (145–2C11) in carbonate buffer overnight at 4°C. Then 106 PP or MLN cells were cultured in 1 ml of complete medium in precoated or uncoated wells. After 72 h, culture supernatants were removed and assayed for cytokine concentration. IL-5, IFN-γ, and TNF-α concentrations were determined by specific ELISA using the manufacturer's protocol (PharMingen).

4.6 Adoptive transfer experiments

Adult MHC-matched SCID mice at 16 weeks of age were the recipients in the adoptive transfer experiments. SAMP1/Yit mice and AKR/J mice were killed at 24 weeks of age to harvest MLN cells. These were positively selected for CD4+ T cells by incubation with anti-CD4-bound magnetic beads, followed by magnetic cell sorting (Miltenyi Biotec, Auburn, CA). SCID mice were injected intraperitoneally with 106 CD4+ T cell-enriched MLN cells. In a subset of experiments, SCID mice were treated with double injections of 30 μg of rat anti-mouse IL-5 antibody (TRFK-5)or rat IgG1 (R3–34) (PharMingen) 2 h before receiving CD4+ T cells and again after 3 weeks. Mice were killed 6 weeks later to remove the small intestine and colon for histological evaluation in a blinded fashion.

4.7 Statistical analysis

Data were expressed as means ± SEM, and statistical analysis was performed using Student's t-test or ANOVA with Turkey's test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This work was supported by the Ministry of Education, Science, Sports and Culture of Japan as part of a project for establishing new high-technology research centers.

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