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Keywords:

  • Th1/Th2 cells;
  • Cytokines;
  • Mucosa;
  • Parasitic helminth

Abstract

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

Expulsion of the gastro-intestinal nematode Trichinella spiralis is associated with a pronounced mastocytosis mediated by a T helper (Th) 2 type response involving interleukin (IL)-4 and IL-13. Here we demonstrate that IL-10 is a key regulator of protective immune responses against T. spiralisin vivo. IL-10 knockout mice or normal mice treated with a neutralizing anti-IL-10 receptor antibody are highly susceptible to a primary T. spiralis infection and show significantly delayed adult worm expulsion. Depletion of IL-10 resulted in elevated Th1 and Th2 cytokine responses but significantly reduced numbers of mucosal mast cells in the jejunum. Interestingly, the increase in IFN-γ detected in the absence of IL-10 resulted in increased immunity to larval stages. Hence, IL-10 has a negative effect on immunity to the tissue dwelling larval stages of T. spiralis but plays a significant biological role as an in vivo regulator of intestinal mast cell responses and is crucially involved in protection against adult stages of intestinal parasites in vivo.

Abbreviations:
MLN:

Mesenteric lymph node

MMC:

Mucosal mast cell

KO:

Knockout

p.i.:

Post infection

MMCP-1:

Mucosal mast cell protease-1

VCU:

Villus crypt unit

WT:

Wild-type

1 Introduction

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

Gastro-intestinal nematodes cause some of the worlds most prevalent and chronic human diseases. CD4+ T helper type 2 (Th2) cells generated in the mesenteric lymph nodes (MLN) during the course of infection are critical in host protective immunity to many intestinal nematodes, including Trichinella spiralis1. The life cycle of T. spiralis involves the production of microscopic newborn larvae that are produced by adult female worms in the small intestinal epithelium. The newborn larvae transverse the mucosal tissues in the intestine and disseminate systemically in the host. The expulsion mechanism of adult T. spiralis worms from the small intestine is a complicated immune-mediated process, which involves the activation of Th2 cells 25 and mucosal mast cells (MMC) 68.

IL-10 is a regulatory cytokine which inhibits both antigen presentation and subsequent pro-inflammatory cytokine release 913. In addition, there is evidence that it promotes formation of antigen-specific regulatory T cell clones 14, 15. The pivotal role played by IL-10 within the mucosal immune system is demonstrated by the chronic colitis that develops in IL-10 knockout (KO) mice, and by its therapeutic efficacy in several animal models of colitis 1618.

In this report we provide new information on IL-10 as a key regulator of Th1 and Th2 responses in the small intestine. To exclude effects of the spontaneous intestinal inflammation that frequently develops in IL-10-deficient mice we have compared our data from IL-10-deficient mice with normal mice treated with a blocking anti-IL-10 receptor antibody in vivo. This study provides conclusive evidence that IL-10 has a dual role as a negative and positive regulator of immune responses directed against the different life stages of parasite infection. Furthermore, IL-10 acts as an early regulator of MMC responses in vivo.

2 Results

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

2.1 IL-10 KO mice are more susceptible to infection with adult T. spiralis than wild-type mice, but develop lower levels of encysted muscle larvae

The response of IL-10 KO mice to primary infections with T. spiralis were examined. Knock out mice and C57Bl/6 wild-type (WT) mice were infected with 300 T. spiralis larvae and worm burdens were assessed at days 8, 12 and 16 post infection (p.i.) (n=5).

WT mice had started expelling the worms by day 12 p.i. and had almost completed the process at day 16 p.i. (Fig. 1A). The IL-10 KO mice, however, had significantly higher worm burdens at both days 12 and 16 p.i. as compared to WT mice (p<0.05) (Fig. 1A). We also investigated the level of fecundity on day 8 p.i. and found no difference between female worms from the different groups (data not shown).

To investigate if the delayed expulsion resulted in higher levels of encysted muscle larvae, we assessed skeletal muscle larvae burdens in the infected groups at 30 days p.i. The results revealed that IL-10 KO mice had almost a 50% decrease in muscle larvae deposition as compared to WT mice (p<0.01) (Fig. 1B). These data clearly demonstrate that the kinetics of adult T. spiralis expulsion from the intestine does not correlate with the number of encysted muscle larvae, and it is likely that different types of immune mechanisms may be involved in protection against the different life stages.

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Figure 1. Delayed expulsion of adult T. spiralis worms from the small intestine of IL-10 KO mice. (A) WT (black bars) and IL-10 KO (white bars) mice on a C57BL/6 background were inoculated orally with 300 T. spiralis muscle larvae. Mice were killed and adult worms were counted at the time points indicated. Values represent mean and SEM, and five to six mice were used per group in this and all subsequent experiments. (B) WT and IL-10 KO mice were inoculated as above and number of muscle larvae determined 30 days later. (C) WT and IL-10 KO mice were inoculated with T. spiralis as above and lengths of jejunum were collected on various days p.i., histologically processed, sectioned, stained with toluidine blue and numbers of MMC per 20 randomly selected VCU were determined by light microscopy. (D) Sera were collected from the same groups of mice as above and analyzed by ELISA for MMCP-1 levels. Asterisks indicate statistically significant difference between KO and WT (p<0.05).

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2.2 IL-10 KO mice exhibit decreased MMC responses during T. spiralis infection

Since mucosal mast cells play an important role in the expulsion of T. spiralis3, 7, 19 we investigated mast cell recruitment and activity in infected mice from the two groups. The number of MMC in the jejunum of IL-10 KO mice were significantly decreased as compared to WT controls at day 8 p.i. (p<0.05) (Fig. 1C). The MMC response in IL-10KO mice then recovered to the same levels as seen in WT controls on days 12 and 16 p.i. (Fig. 1C). There was no significant difference in the number of MMC in the uninfected animals (Fig. 1C).

To determine if the decreased recruitment of mast cells to the small intestine in IL-10 KO mice early during T. spiralis infection was also reflected in decreased mast cell degranulation, we analyzed the levels of MMC protease-1 (MMCP-1) in serum. IL-10 KO mice expressed significantly lower levels of serum MMCP-1 at day 8 p.i. as compared to WT controls (p<0.05, Fig. 1D). The values then increased to similar levels as detected in the WT controls on days 12 and 16 p.i. (Fig. 1D).

We also investigated the number of goblet cells and eosinophils in the small intestine during infection and found no significant difference between the groups (data not shown), demonstrating that the absence of IL-10 preferentially inhibits MMC responses early in infection.

2.3 MLN cells from T. spiralis infected IL-10 KO mice secrete high levels of antigen-specific Th2 cytokines

The cytokines IL-4, IL-13 and IL-10 are known to be of importance in the development and recruitment of MMC 4, 20, 21. To investigate the cytokine response in T. spiralis-infected IL-10 KO and WT mice, MLN cells were harvested at various time points p.i. and re-stimulated in vitro with T. spiralis antigen. The results in Fig. 2 demonstrate that IL-10 KO mice develop elevated Th2 responses as compared with WT mice. IL-10 KO mice displayed a twofold increase in IL-4 secretion as compared to WT mice on day 8 p.i. (IL-10 KO, 8,619±1,704 pg/ml, WT mice, 3,246±185 pg/ml, p<0.03, Fig. 2A), and also a twofold increase in IL-13 secretion (IL-10 KO, 60.32 ±11.86 ng/ml, WT mice, 20.01±2.28 ng/ml, p<0.03, Fig. 2B).

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Figure 2. Decreased resistance to T. spiralis in IL-10 KO mice is associated with increased IL-4, IL-13 and IFN-γ secretion. MLN cells from T. spiralis infected WT (black bars) and IL-10 KO (white bars) mice were removed at various time points during infection and stimulated in vitro with T. spiralis antigen. Supernatants were analyzed by sandwich ELISA for the presence of IL-4 (A), IL-13 (B), and IFN-γ (C). Asterisks indicate statistically significant difference between KO and WT (p<0.05).

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2.4 IL-10 KO mice show elevated Th1 responses during T. spiralis infection

To investigate whether the strong increase in Th2 responses seen in the IL-10 KO mice was reflected in a similar decrease in Th1 response, we measured the levels of IFN-γ. The secretion of IFN-γ was significantly increased in IL-10 KO as compared to WT mice at all time points (p<0.03, Fig. 2C). Thus, these results demonstrate that Th1 and Th2 responses can develop simultaneously during an in vivo infection.

2.5 In vivo neutralization of IL-10 delays T. spiralis expulsion in NIH mice but decrease muscle larvae burdens

Since IL-10 KO mice frequently develop spontaneous intestinal inflammation, which may affect the results in a nonspecific manner, we felt that it was necessary to confirm our results using normal mice treated with a neutralizing antibody in vivo. T. spiralis-infected NIH mice were treated with injections of 1 mg blocking anti-IL-10 receptor antibody on days 3, 6 and 9 p.i. NIH mice are fast responders to T. spiralis infection and have normally completed worm expulsion around days 10–14 p.i. Control NIH mice treated with rat IgG started expelling the worms on day 7 p.i., while the anti-IL-10 receptor-treated group still exhibited full worm burden at this time point (Fig. 3A, p<0.02). At 11 days p.i., the control group had completed expulsion, while the anti-IL-10 receptor-treated animals still had significant worm burdens (Fig. 3A, p<0.01).

To investigate the biological significance of the delayed expulsion observed in the anti-IL-10-treated animals, skeletal muscle larvae burdens were assessed at 30 days p.i. The control group had a 1.6-fold higher number of muscle larvae as compared to the group that received anti-IL-10 receptor treatment (p<0.02), confirming that IL-10 plays an important biological role in the outcome of T. spiralis infection in vivo (Fig. 3B).

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Figure 3. neutralization of IL-10 results in delayed T. spiralis expulsion, but decreased muscle larvae burden in NIH mice. (A) NIH mice were inoculated orally with 300 T. spiralis muscle larvae. One group received rat IgG injections (black bars) and one group were injected with anti-IL-10 receptor antibody (striped bars). Mice were killed and adult worms were counted at the time points indicated. (B) NIH mice were inoculated and injected as above and number of muscle larvae determined 30 days later. (C) Lengths of jejunum were collected from the same groups of mice as above on various days p.i., histologically processed, sectioned, stained with toluidine blue and numbers of MMC per 20 randomly selected VCU were determined by light microscopy. (D) Sera were collected from the same groups of mice as above and analyzed by ELISA for MMCP-1 levels. Asterisks indicate statistically significant difference between groups (p<0.05).

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2.6 Anti-IL-10 receptor treatment inhibits T. spiralis induced mastocytosis in vivo

The numbers of MMC in the jejunum of the anti-IL-10 receptor-treated mice were significantly decreased on days 11 and 15 p.i. as compared to rat IgG-treated controls (p<0.05) (Fig. 3C). There was no significant difference in mast cell numbers at any other time point. Furthermore, the levels of MMCP-1 in serum were significantly decreased in the anti-IL-10 receptor-treated group as compared to controls on day 11 p.i. (p<0.05) (Fig. 3D). Taken together these data demonstrate that in vivo treatment with anti-IL-10 receptor antibody inhibits MMC recruitment as well as maturation and/or activation.

2.7 In vivo treatment with anti-IL-10 receptor increases lymph node secretion of IL-4, IL-13 and IFN-γ 

When cytokine secretion from antigen-stimulated MLN cultures were examined, the results demonstrate that the rat IgG-treated NIH mice developed strong Th2 responses during the course of T. spiralis infection (Fig. 4). The anti-IL-10 receptor-treated NIH mice however, had significantly increased secretion of IL-4 on days 4 and 7 p.i. (p<0.02 on day 4 and p<0.04 on day 7, Fig. 4A) and IL-13 on day 4 p.i. (p<0.04, Fig. 4B). Antigen-specific IFN-γ secretion was significantly increased in the anti-IL-10 receptor-treated group at all time points (Fig. 4C).

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Figure 4. neutralization of IL-10 results in increased IL-4, IL-13 and IFN-γ secretion. MLN cells from control (black bars) or anti-IL-10 receptor (striped bars) treated T. spiralis infected NIH mice were removed at various time points during infection and stimulated in vitro with T. spiralis antigen. Supernatants were analyzed by sandwich ELISA for the presence of IL-4 (A), IL-13 (B) and IFN-γ (C). Asterisks indicate statistically significant difference between groups (p<0.05).

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2.8 In vivo neutralization of IFN-γ in IL-10 KO mice does not alter T. spiralis expulsion but increase muscle larvae burdens

Our data demonstrate that the removal of IL-10 results in an inability to expel adult worms. It also shows that the absence of IL-10 results in increased immunity against newborn larvae and reduced levels of muscle larvae cysts. These effects on the parasitological parameters were correlated with an increased IFN-γ secretion. To investigate the role of IFN-γ, we treated IL-10 KO mice with injections of a neutralizing anti-IFN-γ antibody on days 2, 4, 6, 8 and 10 p.i.

The data shown in Fig. 5A reveal that in vivo neutralization of IFN-γ in T. spiralis-infected IL-10 KO mice did not alter the rate of expulsion of adult worms from the small intestine, thus demonstrating that the inability to expel the worms was not due to the high levels of IFN-γ produced in the absence of IL-10. However, when IFN-γ was neutralized in vivo the levels of encysted muscle larvae increased significantly (Fig. 5B), indicating that IFN-γ is responsible for the reduced levels of muscle larvae seen in IL-10 KO animals. Moreover, when T. spiralis-infected C57BL/6 and NIH mice were treated with anti-IFN-γ antibody in vivo, we could again detect a significant increase in the number of encysted muscle larvae (data not shown), demonstrating that IFN-γ has a significant biological role in preventing cyst formation in vivo.

The neutralization of IFN-γ in IL-10 KO mice had little or no effect on mast cell or cytokine responses during T. spiralis infection (data not shown).

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Figure 5. The increased production of IFN-γ in IL-10 KO mice is responsible for the decrease in muscle larvae deposition. (A) IL-10 KO mice were inoculated orally with 300 T. spiralis muscle larvae. One group received rat IgG injections (black bars) and one group were injected with a neutralizing anti-IFN-γ antibody (hatched bars). Mice were killed and adult worms were counted at the time points indicated. (B) IL-10 KO mice were inoculated and injected as above and number of muscle larvae determined 30 days later. Asterisk indicates statistically significant difference between groups (p<0.05).

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3 Discussion

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

We have previously reported that pro-inflammatory cytokines, such as IL-18, IL-12 and IFN-γ are crucial regulators of Th2-mediated immunity to intestinal helminth infection 5, 22. In this study we have used normal and IL-10 KO mice to examine the interplay of IL-10 with other cytokines during the in vivo priming of an immune response leading to a polarized Th cell response. The data presented here provide evidence that IL-10 regulates the development of mast cell and cytokine responses in the small intestine, and that the balance between IL-10 and IFN-γ determines the development of immunity against the different life stages of the parasite.

IL-10 was initially characterized as a factor generated by Th2 cells that prevents cytokine production by Th1 cells 9. However, several other cell types have been further identified as a source of this cytokine, including other T cell subsets, mast cells, B cells, epithelial cells and particularly macrophages 23, 24. The effects of IL-10 are reported to be mainly anti-inflammatory and includes inhibition of cytokines such as IL-1, IL-6, IL-8 and TNF-α 23, 24. Since these cytokines play a critical role in the activation and recruitment of various cell types to the site of inflammation, it suggests that IL-10 plays a pivotal role in orchestrating the inflammatory reaction. IL-10 is also an important regulatory cytokine within the mucosal immune system as demonstrated by the chronic colitis that develops in IL-10 KO mice, and by its therapeutic efficacy in several animal models of colitis 1618.

Mouse strains can be divided into "slow" or "fast" responders according to their ability to expel T. spiralis from the small intestine 19, 25. The speed of expulsion is clearly correlated with the capability of developing MMC hyperplasia 19, and blocking mast cell development by anti-stem cell factor treatment or by blocking the c-kit receptor in vivo significantly delays worm expulsion 6, 7. The exact mechanism of expulsion is not clear, but is known to be dependent on a Th2 type of response (involving IL-4, IL-13 and IL-9), which leads to the activation of MMC 1, 3, 4, 6, 7, 26.

Since IL-10 inhibits IFN-γ secretion we wanted to investigate the importance of IL-10 during a T. spiralis infection in vivo. When IL-10 KO and WT (C57BL/6) mice were infected with T. spiralis, the IL-10 KO mice displayed severely impaired expulsion rate of the adult worms. Although our IL-10 KO mice were kept under SPF conditions, used at a young age (5–7 weeks old at the start of the experiments) and did not display any symptoms of intestinal inflammation, we felt it was important to confirm our findings in a normal mouse strain. We used NIH mice that are "fast responders" in terms of T. spiralis expulsion (the expulsion process is usually completed around days 10–12 p.i.) 19, 25. When NIH mice were treated with injections of a neutralizing anti-IL-10 receptor antibody 27, they also showed significantly delayed adult worm expulsion. This finding, together with the IL-10 KO data, confirm that depletion of IL-10, even in a "fast responder" strain such as NIH, will result in significantly delayed worm expulsion. Thus, IL-10 is crucial for the expulsion of adult T. spiralis worms from the small intestine in vivo.

When we investigated the cytokine basis for the observed differences in the kinetics of worm expulsion, the results showed that the delayed expulsion seen in IL-10 KO mice as well as in anti-IL-10 receptor-treated NIH mice correlated well with high levels of antigen-specific IFN-γ secretion. This is in line with previous reports establishing that IL-10 deficiency results in increased IFN-γ responses in vivo (reviewed in 24). Interestingly, when we measured antigen-specific Th2 responses we found significantly elevated levels of both IL-4 and IL-13 being produced in the infected IL-10 KO mice as well as in the anti-IL-10 receptor-treated NIH mice as compared to the WT controls. Originally, it was believed that Th2 and Th1 responses would antagonize and thereby ameliorate each other's consequences, but our data demonstrate that strong Th1 and Th2 responses can develop simultaneously in vivo and that the development of one type of Th cell response does not necessary inhibit or prevent the development of the other. The finding of mixed Th1 and Th2 responses have previously been reported in lung inflammation models 28, 29 and in a model of allergic responses to Aspergillus antigen the deletion of IL-10 resulted in exaggerated Th1 and Th2 responses in the lungs 30. Thus, our data confirm that IL-10 may regulate both Th1 and Th2 responses at mucosal surfaces.

The maturation and development of MMC are largely dependent on Th2 cytokines such as IL-3, IL-4, IL-13, IL-9 4, 20, 31 and, in particular, IL-10 21, 3237. IL-10 alone has little ability to promote mast cell growth, but when used in combination with IL-3, IL-4 or stem cell factor induces proliferation of MMC. IL-10 also induces expression of mast cell proteases and is reported to be more potent than IL-4 in inducing maturation. Both T. spiralis-infected IL-10 KO mice and the anti-IL-10 receptor-treated NIH mice showed significant delayed or reduced development of jejunal mastocytosis during infection, correlating with the slow expulsion rate of the parasite. Goblet cell hyperplasia and the development of intestinal eosinophilia was not affected by IL-10 deficiency or neutralization, demonstrating that the effect of IL-10 appears to be specific for MMC in vivo. It is clear from our data that IL-10 is an absolute requirement for the fast development of a jejunal mast cell response in vivo, since a delayed mast cell response was detected in the absence of IL-10, although the levels of IL-4 and IL-13 were elevated. The level of MMCP-1 in serum, a marker for mature mast cells, was also reduced or delayed, thus confirming the role for IL-10 in promoting mast cell maturation in vivo.

The life cycle of T. spiralis involves the production of microscopic newborn larvae that are produced by adult female worms in the small intestinal wall. The newborn larvae transversethe mucosal tissues in the intestine and disseminate systemically in the host. Each stage of the T. spiralis life cycle can evoke a protective host immune response and each response is stage specific due to the expression of unique cuticular and excretory-secretory antigens at each stage 3844. Our data confirm that different mechanisms of immunityoperate against adult worms and newborn larvae, and that this immunity is regulated, at least in part, by IL-10. Although IL-10 KO and anti-IL-10 receptor-treated mice showed significantly delayed expulsion of adult worms from the intestine, this was not reflected in an increase in the number of encysted muscle larvae. In fact, the numbers of encysted muscle larvae were significantly decreased in IL-10-deficient or -depleted animals, demonstrating that other mechanisms of protective immunity are operating against newborn larvae, and that these mechanisms are distinct from those involved in expulsion of adult worms. Furthermore, this immunity to newborn larvae involves the production of IFN-γ since in vivo neutralization of IFN-γ in IL-10 KO mice increased the levels ofencysted muscle larvae. However, this treatment did not alter the kinetics of adult worm expulsion, demonstrating that the increased levels of IFN-γ seen in IL-10 KO mice are not responsible for the delay in worm expulsion. In summary, the data presented here demonstrate that IFN-γ is crucially involved in protection against newborn larvae, but does not affect the expulsion of adult worms. Mechanisms of IFN-γ-mediated immunity to newborn larvae may include enhanced cytotoxic killing by eosinophils, granulocytes and activated macrophages 4549.

In this report we provide new information on IL-10 as a key regulator of MMC development and cytokine responses in the small intestine. This study provides, for the first time, conclusive evidence that IL-10 is a crucial mediator of resistance to T. spiralisin vivo, but that the absence of IL-10 allows IFN-γ to exert its protective effects against newborn larvae. Thus, IL-10 function as a double-edged sword in both promoting immunity and pathology associated with intestinal nematode infection.

4 Material and methods

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

4.1 Animals and infections

Male NIH and C57BL/6 mice, 6–8 week old, were purchased from Harlan Olac Ltd. (Bicester, GB). IL-10 KO mice on a C57BL/6 background 16 were bred at the animal unit, The University of Manchester under SPF conditions. All experiments were performed under the regulations of the Home Office Scientific Procedures Act (1986).

Maintenance, infection and recovery of T. spiralis were as described previously 50. Experimental mice were infected with 300 infective T. spiralis larvae by oral gavage on day 0, and the numbers of adult worms in the small intestine were assessed at various time points p.i., as detailed in the text. Muscle larvae burden were determined on day 30 p.i. T. spiralis antigen was prepared as described previously 1. Measurements of adult female worm fecundity was performed by placing individual female worms in 100 μl PBScontaining 10% heat-inactivated FCS (Invitrogen, Paisley, GB) in 96-well plates. After a 5-h incubation at 37°C the numbers of newborn larvae shed from each female worm were counted using an inverted microscope.

In vivo neutralization with anti-IL-10R antibody (1B1.3a, 27) was performed by i.p. injections of 1 mg antibody per mouse on days 3, 6 and 9 p.i. In vivo neutralization with anti-IFN-γ antibody (XMG1.6) was performed by i.p. injections of 1 mg antibody per mouse on days 2, 4, 6 and 10 p.i. Control mice received i.p. injections of rat IgG (Sigma, Gillingham, GB).

4.2 Cell culture and cytokine analysis

MLN cells were removed from uninfected and infected animals and resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.05 mM 2-mercaptoethanol (all from Invitrogen, Paisley, GB). MLN were cultured at 37°C and 5% CO2 in flat-bottom 96-well plates (Nunc, Roskilde, Denmark) at a final concentration of 5×106/ml in a final volume of 0.2 ml/well. Cells were stimulated with T. spiralis antigen (50 μg/ml). Anti-IL-4 receptor mAb (M1, 5 μg/ml: from Dr. C. Maliszewski, Immunex Corp. Seattle, WA) was added to cultures to increase detection of IL-4. Cell-free supernatants were harvested after 48 h and stored at –80°C.

4.3 Cytokine ELISA

Cytokine analyses were carried out using sandwich ELISA for IL-4 (mAb 11B11 and BVD6–24G2: Mabtech AB, Nacka, Sweden) and IFN-γ (AN18 and R46A2, Mabtech AB). IL-13 and IL-10 were analyzed using antibody pairs from R&D systems (Abingdon, GB).

4.4 MMCP-1 analysis

Serum levels of MMCP-1 were determined using a commercially available kit (Moredun Animal Health Ltd, Penicuik, GB).

4.5 Histology

Consecutive lengths of small intestine taken 10 cm from the pyloric sphincter were fixed in Carnoys fluid or neutral buffered formalin, histologically processed using standard methods, and 5-μm sections were stained for MMC (0.5% toluidine blue), goblet cells (periodic acid-Schiff) and eosinophils (hematoxylin-eosin). The number of cells per 20 randomly selected villus-crypt units (VCU) were determined under light microscopy from at least two sections per animal.

4.6 Statistics

Significant differences (p<0.05) between experimental groups were determined using the Mann-Whitney U test.

Acknowledgements

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

We would like to thank R. L. Coffman, DNAX Research Institute of Molecular and Cellular Biology, for providing the anti-IL-10 receptor antibody. We would also like to thank Ms. Ann Lowe for support with histology. This work was supported by the Wellcome Trust.

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