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Keywords:

  • Dendritic cells;
  • Intestinal mucosa;
  • Lamina propria;
  • Oral tolerance

Abstract

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

The lamina propria (LP) of the small intestine contains many dendritic cells (DC), which are likely to be in close contact with luminal antigens, but their role in intestinal immune responses has been overlooked. Here we show that after feeding mice ovalbumin (OVA), the majority of antigen uptake is associated with DC in the small intestinal LP, and we describe the isolation, purification and initial characterization of theses DC. We obtained >90% CD11c+ DC using magnetic cell sorting, of which the majority were CD11b+CD8α, with smaller numbers of CD11bCD8α+ and CD11bCD8α DC as well as a distinct population of CD11cintclass II MHClo B220+ DC. Freshly isolated LP DC expressed variable but generally low levels of CD40, CD80 and CD86, which were up-regulated by activation with LPS. LP DC were endocytic in vivo and in vitro and could present antigen to OVA-specific CD4+ T cells in vitro. Antigen-loaded LP DC from OVA-fed mice also primed specific CD4+ T cells in vivo and in vitro, but adoptive transfer of these DC into naive recipients induced hyporesponsiveness to subsequent challenge. LP DC also expressed significant levels of mRNA for IL-10 and type I IFN, but not IL-12, suggesting they may play a central and unique role in immune homeostasis in the gut.

Abbreviations:
LP:

Lamina propria

PP:

Peyer's patches

MLN:

Mesenteric lymph nodes

DTH:

Delayed-type hypersensitivity

Introduction

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

Oral administration of protein antigens induces systemic immunological tolerance, and this is believed to be the physiological process that prevents food hypersensitivity reactions such as food allergy and celiac disease 1, 2. A similar process directed at commensal bacteria in the large intestine may prevent inflammatory bowel diseases such as Crohn's disease. It is clear from other systems that the way in which antigen is presented to the immune system is the critical factor that controls whether tolerance or immunity results, and most evidence indicates that DC are likely to be the APC involved. In the presence of inflammation or pathogenic organisms, DC are activated to express a full range of costimulatory molecules and cytokines, ensuring the efficient stimulation and differentiation of effector T cells 3. However, DC are also central to the induction of tolerance to both self and foreign antigens, as in the absence of inflammation, DC can present antigen to T cells but lack the complete range of costimulatory molecules necessary for full T cell activation. DC of this kind have been referred to as mature but "quiescent", and T cells stimulated in this way become unresponsive (anergic) and/or differentiate into regulatory T cells 3.

We showed previously that DC are important for the induction of oral tolerance, as the expansion of DC numbers in vivo using the cytokine flt3 ligand enhances the susceptibility of mice to the induction of tolerance by feeding OVA 4. Conventionally it is assumed that the APC involved in the uptake and presentation of intestinal antigens are in Peyer's patches (PP) 5, and the PP contain unusual subsets of DC, some of which can produce IL-10 and drive the production of IL-10 by antigen-specific T cells 6. These findings therefore support the idea that PP DC may be important for the generation of Treg and oral tolerance. However, studies using mice deficient in PP have produced conflicting evidence concerning the requirement of these organs for the induction of oral tolerance 710. Thus we considered the possibility that DC in another site might be more important for the uptake and presentation of orally administered protein antigens.

The conventional villus epithelium of the small intestinal mucosa represents a much greater surface area than that of PP and can allow transport of macromolecules such as intact proteins 11. It has long been known that DC are present in the lamina propria (LP) underlying the villus epithelium 12, and it has been suggested that some of these cells may be able to migrate into the epithelium 13. Despite the evidence that DC in the LP may be in an ideal position to take up protein antigens from the intestine, little is known of their properties or of their role in intestinal immunoregulation. In this study, we have developed techniques for purifying DC from mouse small intestine and have assessed the characteristics and functions of these cells. Our results show that DC in the LP are loaded very efficiently by feeding OVA to mice, and several subsets of DC can be purified from this site. LP DC can present antigen to antigen-specific CD4+ T cells in vivo and in vitro, but when transferred into normal recipients, they appear to induce a state of immune hyporesponsiveness. In parallel, LP DC produce more mRNA for IL-10 and type 1 IFN but less IL-12 mRNA than do spleen DC, suggesting that there may be a population of DC in LP that can preferentially induce T cell tolerance after the feeding of antigen.

Results

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

Phenotypic characterization of DC in intestinal LP

CD11c+ cells with the appearance of DC are present in the normal LP and many are located immediately below the epithelium, in an ideal position to sample gut antigen (Fig. 1a). To characterize these DC, we isolated mononuclear cells from the LP of normal mice and analyzed them by flow cytometry. Normal LP preparations contained 4% CD11c+ cells of which approximately 60% were CD11b+CD8α, 20% were CD11bCD8α+, and the remainder were CD11bCD8α (Fig. 1b and Table 1). In comparison with other tissues, LP were more similar to PP than spleen, with the majority of DC being CD11b+CD8α and there was a significant population of CD11bCD8α DC (Table 1 and 14, 15). The majority of CD11c+ cells from the LP were Class II MHCint/hi, confirming them as relatively mature DC.

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Figure 1. Analysis of DC in small intestinal LP. a) DC are present in the LP of the jejunum of normal mice, and their numbers are expanded after treatment of mice with flt3L. Confocal microscopy images of intestine stained with DAPI to demonstrate cell nuclei (blue) and with anti-CD11c visualized with Alexa Fluor 647 (red) to detect DC are shown (magnification ×100). b) Proportions of CD11c+ cells among lymphoid cells isolated from normal LP, from the LP of flt3L-treated mice and after positive selection of CD11c+ cells from the LP of flt3L-treated mice, as assessed by flow cytometry. The expression of CD11b and CD8α on live-gated (PI) CD11c+ cells from normal LP, flt3L-treated LP and among purified DC from flt3L-treated LP is also shown. The data are representative of at least four similar experiments.

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Table 1. Phenotype of DC from LP, spleen and PPa)
Lamina propriaSpleenPeyer's patches
  1. a) Proportions of CD11c+ DC expressing different subset markers in samples of freshly isolated mononuclear cells assessed by flow cytometry. The data shown are the percentage of live-gated (PI) CD11c+ cells expressing each marker and are the means ± one SD of three separate experiments.

MHC class II+75.8±12.6%58.6±6.9%59.2±7.6%
CD11b+48.8±5.7%46.1±4.7%34.9±5.4%
CD8α+17.8±3.5%23.1±4.8%12.7±2.3%
CD40+13.3±3.1%8.0±3.0%20.1±2.3%
CD80+44.5±13.8%33.0±5.1%18.2±2.1%
CD86+30.1±9.6%15.5±6.2%6.9±0.6%

To characterize LP DC in more detail and to purify these cells, we treated mice with flt3L to increase the yields of DC. This increased the proportion of CD11c+ cells in the LP from 4% to 25±5% (Fig. 1a), without significantly altering the relative proportions of the major subsets of CD11b+ or CD8α+ DC, although there was some increase in the level of CD8 staining on CD8+ DC from flt3L-treated mice (Fig. 1b). These data confirm our previous findings that flt3L-recruited DC reflect all the DC populations found in the normal intestine 4.

Using magnetic purification, we could obtain 1×105 to 5×105 CD11c+ cells per gut with a purity of 80%–92% and without affecting the proportion of CD8α+, CD11b+ or CD8αCD11b DC (means of 24%, 63% and 15% in two experiments). As some CD8α+ intraepithelial lymphocytes in mice have been described to express low levels of CD11c 16, we examined the numbers of contaminating T lymphocytes in our preparations. Less than 2% of the cells were CD3+, indicating that the population of CD11c+CD8α+ cells we had isolated were not contaminating intraepithelial lymphocytes (IEL) and were indeed DC (data not shown). The increased numbers of cells available for analysis now revealed that approximately 15% of DC in LP express intermediate levels of CD11c, the majority of which are also class II MHClow (R3 in Fig. 2). Approximately 30% of these CD11cintclassII MHClow DC were B220+Ly-6C/G+, and the remainder were B220Ly-6C/G (Fig. 2). In contrast, CD11chiclassII MHChi DC (R4 in Fig. 2) were uniformly B220Ly-6C/G. A small number of the CD11cintclassII MHClow DC were also CD8α+, and this proportion was greater than that among the CD11chiclassII MHChi subset (Fig. 2).

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Figure 2. Phenotypic analysis of CD11cint DC from the small intestinal LP of flt3L-treated mice. Unpurified LP cells contained a population of CD11cint class II MHClo DC (R3), which were less numerous than conventional CD11chi class II MHChi DC (R4). After purification, CD11c+ DC were analyzed for co-expression of CD11b, CD8α, B220, Ly6C/G and class II MHC by flow cytometry. The histograms show the levels of expression of each of these markers on CD11clo (R3, thin line) and CD11chi (R4, bold line) DC. The data are representative of at least four similar experiments.

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Phenotypic response of LP DC to activation

We next examined the activation status of LP DC directly ex vivo and after stimulation with LPS. Freshly isolated DC from LP showed variable but generally low levels of CD40, CD80 and CD86 expression comparable with the levels found on DC isolated from the spleen or PP (Fig. 3). LP DC that were cultured in medium overnight showed an increase in the expression of class II MHC, CD80, CD86 and CD40, and this was further enhanced by stimulation with LPS. These phenotypic changes were similar to or greater than those seen when purified splenic DC were activated in the same ways (Fig. 3), indicating that LP DC are not fully mature in situ but are capable of differentiating further in response to appropriate stimuli.

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Figure 3. LP DC are resting in situ but can be activated. Flow cytometry was used to analyze the expression of class II MHC, CD40, CD80 and CD86 on CD11c+ DC from LP and spleen that were freshly isolated (dotted line) or had been cultured overnight in medium (thin line) or with 10 μg/ml LPS (bold line). The data are representative of at least three similar experiments.

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LP DC are capable of taking up orally administered macromolecules

We next examined the uptake of macromolecules by LP DC in vitro and in vivo. DC purified from LP, PP and spleen showed equivalent abilities to endocytose FITC-dextran in vitro, confirming the presence of relatively immature DC in all of these sites (Fig. 4a). After oral administration of FITC-dextran, labeled DC could be identified in the mucosal LP and PP, with similar kinetics of uptake and the peak uptake occurring at 60 min. DC isolated from PP showed higher levels of uptake at each time point (Fig. 4b). DC from mesenteric lymph nodes (MLN) and spleen showed no uptake of orally administered FITC-dextran (Fig. 4b and data not shown).

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Figure 4. Endocytic activity of LP DC in vitro and in vivo. a) LP (squares), spleen (triangles) and PP (circles) cells from flt3L-treated mice were incubated with 1 mg/ml FITC-dextran for 5–60 min at 37°C before being stained with PE-anti-CD11c. The uptake of FITC-dextran was assessed by analysis of live-gated (PI) CD11c+ DC. The results shown are the mean ΔMFI ± one SD of three experiments using duplicate cultures. b) Flt3L-treated mice were fed 5 mg FITC-dextran, and LP (squares), MLN (triangles) and PP (circles) cells were isolated 15–180 min later before being stained with PE-anti-CD11c. The results show the uptake of FITC by live CD11c+ DC and are the mean ΔMFI ± one SD of three experiments using duplicate cultures, calculated by subtracting the MFI obtained when the equivalent cells were isolated from saline-fed mice.

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APC activity of LP DC

To investigate the APC function of LP DC, we examined their ability to present antigens to T cells in vitro and in vivo. DC purified from LP, PP or spleen were pulsed with OVA in vitro and then used to stimulate DO11.10 T cells. DC from all tissues were able to induce significant proliferation of T cells in vitro, although DC from LP and PP appeared somewhat less efficient at stimulating T cells than splenic DC at all T cell:APC ratios examined (Fig. 5a). Antigen-loaded LP DC purified from mice fed OVA 1 h before induced strong proliferation of DO11.10 T cells in vitro, which was similar to or even greater than that induced by the same numbers of DC purified from the PP of the same mice (Fig. 5b). Together these results show that DC in the LP possess a high ability to take up soluble antigens from the gut lumen.

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Figure 5. Antigen presentation activity of DC from intestinal LP in vitro. a) CD11c+ DC from LP (open bars), PP (shaded bars) and spleen (filled bars) were purified, pulsed with 10 mg/ml OVA for 2 h, treated with mitomycin c and used at the DC numbers indicated to stimulate 2×105 DO11.10 TCR-transgenic lymphocytes for 72 h in vitro. b) CD11c+ DC were purified from the LP (open bars) and PP (shaded bars) of mice fed 200 mg OVA 1 h before. The DC were then treated with mitomycin c and used to stimulate DO11.10 lymphocytes as above. The cultures were pulsed with 1 μCi/ml [3H]-TdR/well for the last 18 h of culture, and the results shown are the mean cpm ± one SD for triplicate cultures. The data are representative of three similar experiments.

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We next explored the idea that LP DC that have taken up fed proteins could contribute to the induction of oral tolerance in vivo. To do this, DC were purified from the LP of mice fed OVA 1 h before sacrifice and were transferred into mice that had already received CFSE-labeled DO11.10 cells. The expansion and activation of the OVA-specific T cells were then assessed in the draining lymph nodes by flow cytometry. As expected, there was clonal expansion of DO11.10 cells in control recipients immunized with OVA/CFA, and the majority of specific T cells expressed CD69 (Fig. 6a) and underwent cell division as determined by loss of CFSE (Fig. 6b). Transfer of in vivo-loaded LP DC also induced specific CD4+ T cell activation in vivo, with 90% of the specific T cells expressing CD69 and approximately 80% having divided by day 7 (Fig. 6a, b). Similar results were obtained using purified splenic DC that had been loaded with OVA in vitro. PP DC from OVA-fed mice also induced antigen-specific T cell activation in vivo but were less efficient than LP DC, with a maximum of 40% of the CD4+KJ1.26+ cells having divided by day 7 (Fig. 6b).

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Figure 6. In situ-loaded LP DC present antigen to CD4+ T cells in vivo but may induce systemic hyporesponsiveness. DC were purified from the LP or PP of mice fed 200 mg OVA 1 h before and were transferred s.c. into mice that had received 3×106 CFSE-labeled KJ1.26+ DO11.10 lymphocytes 24 h before. a) Expression of CD69 on KJ1.26+CD4+ T cells and b) cell division of OVA-specific T cells, expressed as the proportions of KJ1.26+CD4+ cells that have divided since transfer, as indicated by loss of CFSE expression (symbols in a and b correspond to those in c). c) Systemic DTH responses in recipients challenged s.c. with 10 μg OVA/CFA 10 days after transfer of DC. DTH responses were assessed 21 days after challenge, and the results shown are mean increments in footpad thickness for individual mice 24 h after challenge with 100 μg heat-aggregated OVA. Controls received DC from saline-fed donors or DC purified from normal spleens that had been pulsed with OVA in vitro. d) Production of IFN-γ by lymphocytes from the recipients of spleen, LP or PP DC from OVA-fed mice. The cells were taken from the draining lymph nodes 10 days after challenge of the mice with OVA/CFA and were restimulated with OVA in vitro. Results are the mean IFN-γ levels (ng/ml) ± one SD from triplicate cultures (**p<0.01 vs. saline-fed LP DC, unpulsed splenic DC and immunized controls that had not received DC; p⩽0.05 vs. OVA-pulsed spleen DC and OVA-fed PP DC; ***p<0.001 vs. IFN-γ levels in all other groups). The results are representative of three individual experiments.

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When adoptively transferred recipients were challenged with OVA/CFA, mice previously given LP DC from OVA-fed mice had significantly reduced delayed-type hypersensitivity (DTH) responses compared with those seen in control mice that had only been immunized with OVA/CFA (Fig. 6c). Draining lymph node lymphocytes from these mice also showed significantly suppressed production of IFN-γ when restimulated with OVA in vitro compared with cells from immunized controls (Fig. 6d). Mice that received DC purified from the spleen or PP had no evidence of down-regulation of DTH responses and IFN-γ production after challenge with OVA, irrespective of whether they had been loaded with OVA or not (Fig. 6c, d). Mice that received DC purified from the LP of saline-fed mice also had normal systemic immune responses, indicating that the tolerance was antigen-specific. No antigen-specific IL-4, IL-5 or IL-10 production could be detected in any group of recipients (data not shown). Thus, DC from the LP of antigen-fed mice are capable of presenting this antigen to specific CD4+ T cells in vitro and in vivo, but this may lead to tolerance to subsequent challenge in vivo.

LP DC exhibit a unique cytokine profile

As we found that presentation of antigen by DC from LP may have distinctive functional consequences for antigen-specific T cells, we examined factors that might account for this polarization of T cell function. Real-time PCR showed that freshly purified splenic DC express relatively low levels of mRNA encoding IL-12 p40, type I IFN and IL-10, but these cytokines were markedly up-regulated by overnight LPS stimulation, with the exception of type I IFN (Fig. 7). PP DC expressed small amounts of IL-12 p40 mRNA constitutively, and this was enhanced by LPS. No production of type I IFN or IL-10 could be detected by PP DC under either condition. Conversely, LP DC expressed mRNA for type I IFN and IL-10 constitutively, and this was not altered by LPS, whereas IL-12 p40 mRNA levels remained low even after LPS treatment (Fig. 7). Thus, the immunomodulatory effects of LP DC in vivo may be associated with the constitutive production of potentially immunoregulatory cytokines in vitro.

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Figure 7. Expression of cytokine mRNA by LP DC. DC were purified from LP, PP and spleen of flt3L-treated mice and mRNA levels for IL-12p40 (A), IL-10 (B) and IFN-β (C) quantified by real-time PCR using freshly isolated DC (open bars) and DC that had been activated with 10 μg/ml LPS overnight (filled bars). The results shown are the relative amount of mRNA for each cytokine normalized to the amount of HPRT mRNA in each sample. Identical results were obtained in a repeat experiment.

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Discussion

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

It has long been uncertain where soluble antigens are taken up in the gut and how and where they are presented to T cells. There are many APC such as DC in the mucosal LP, and we found that a substantial amount of orally administered protein antigen is taken up by APC in the LP, confirming previous findings of antigen loading at this site 17. We now show directly that DC are likely to be responsible for this phenomenon and have characterized this population for the first time. As in other, non-lymphoid tissues, the majority of LP DC are of the CD11b+CD8α subset, confirming previous preliminary findings in flt3L-treated mice 4. CD11bCD8α+ and CD11bCD8α DC were also present but were less frequent, each accounting for 15–20% of the DC. Overall, this distribution of subsets is similar to what we and others have described in PP, especially with respect to the CD11bCD8α subset, which is more represented than in non-mucosal sites 14, 15. Recent studies have confirmed the presence of CD11bCD8α DC in normal ileal LP 18. In addition, we consistently observed a population of LP DC that expresses low levels of CD11c and class II MHC. These cells were CD11bloCD8α+/–, and some were B220+ and/or Ly6C/G+. Other work has identified analogous populations of CD11clowclass II MHClow DC in other murine tissues, with the B220+Ly6C/G+ subset considered to be the murine equivalent of the type 1 IFN-producing plasmacytoid DC in humans and the B220Ly6C/G subset believed to be a distinct, tolerogenic population 19, 20. Interestingly, recent studies have also identified a tolerogenic population of class II MHCloCD8α+ cells in PP and MLN 21, 22. As others have shown with DC from human colon 23, freshly isolated LP DC from the mouse small intestine were in a relatively resting state, as indicated by their expression of low levels of CD80, CD86 and CD40. However, the expression of these markers was often somewhat higher after magnetic purification, presumably reflecting the mechanical and other stresses of the procedure. In addition, all these markers were readily induced by treatment of LP DC with LPS or even by overnight culture in medium. Indeed, LP DC were generally more responsive to these stimuli than DC from other tissues, perhaps indicating that they have already been partially activated in the local microenvironment. Interestingly, this observation applied to both the CD11chi and CD11cint subsets of LP DC, which up-regulated costimulatory molecules equally well in response to LPS, contrasting with what has been reported recently with CD11clo DC from spleen 19. Together, these results suggest that LP DC are in a resting or "quiescent" state in situ but are potentially responsive to exogenous stimuli.

DC isolated from the LP were capable of endocytosis of FITC-dextran in vitro, with activities generally similar to DC from other sites. In addition, they could take up orally administered FITC-dextran from the intestinal lumen and were capable of APC activity when pulsed with intact antigen in vitro, although this was generally lower than that of splenic DC, perhaps again reflecting the possibility that LP DC have partially matured during isolation, with a resultant decrease in antigen uptake. In vivo-loaded LP DC were also able to present antigen to adoptively transferred antigen-specific CD4+ T cells in vivo, and LP DC from OVA-fed mice were more efficient than PP DC in activating CD4+ T cells in vivo and in vitro. However, the recipients had reduced DTH responses and antigen-specific IFN-γ production compared with controls when challenged subsequently with antigen in adjuvant. This pattern was not seen with other sources of DC, irrespective of whether they had been loaded with antigen in vivo or in vitro. Thus, the outcome of T cell stimulation by LP DC is qualitatively different from that induced by other populations of DC and may play a crucial role in initiating tolerance to intestinal antigens.

In contrast to DC from peripheral tissues, LP DC constitutively expressed cytokines such as IL-10 and type 1 IFN, which have been implicated in the induction and/or maintenance of regulatory T cells 19, 24, 25. In parallel, they did not readily produce cytokines that are more usually associated with active inflammatory responses, even when activated with appropriate stimuli that induce phenotypic maturation of LP DC. We conclude that LP DC are "quiescent" in situ and functionally resistant to inflammatory signals, characteristics that are somewhat paradoxical given their continuous exposure to a wide variety of antigens and other immunomodulatory agents derived from the intestinal lumen. One possible explanation for this could be that DC in the intestinal mucosa represent a distinct lineage, with a specialized and hard-wired role in the induction of tolerance. However, we believe this is unlikely given the increasing evidence that most if not all DC are extremely plastic in nature and are not predestined to fixed functional roles 3, 26. That this also applies to the intestine is suggested by the phenotypic changes we observed when LP DC were stimulated with LPS in vitro and by previous studies showing that mucosal DC may mediate the induction of either tolerance or active immunity depending on the presence of inflammatory signals such as IL-1 or cholera toxin 27, 28. Therefore it is more probable that mucosal DC are derived from a common DC precursor but are maintained in a quiescent state by factors such as PGE2 or TGFβ in the local microenvironment.

Taken together, our results are consistent with the idea that the uptake of antigens by DC in the intestinal LP plays a central role in the induction of oral tolerance to dietary proteins. This is consistent with recent work on other mucosal tissues in which it has been reported that under physiological conditions, DC from the PP, MLN and lung preferentially produce IL-10 and polarize antigen-specific T cells to a Th2 or Treg phenotype in vitro9, 21, 29, 30, 31. In our system, we were unable to detect IL-10 production by T cells taken from mice transferred with any source of DC from OVA-fed mice, and it would be important to investigate this further, e.g. by using additional rounds of restimulation of the T cells. We propose that most intestinal antigens may induce tolerance as a default response because they are taken up and presented by mucosal DC that are geared to tolerize antigen-specific CD4+ T cells. We suggest that LP DC are critical to these processes and that our findings help explain the conflicting evidence on whether PP are essential for the induction of oral tolerance 5. We propose that LP DC will migrate to the draining MLN before interacting with naive CD4+ T cells. This is consistent with both the presence of antigen-loaded DC in the draining mesenteric lymph of antigen-fed rats and mice 9, 29, 31 and the fact that MLN are absolutely required for the induction of oral tolerance 7, 8. That this is a constitutive process is suggested by the large output of DC in lymph draining the intestine under physiological conditions 32 and by the fact that T cell tolerance to self antigens in the periphery appears to be maintained by the homeostatic recirculation of resting, antigen-bearing DC from tissues to lymph nodes 3335. Thus, quiescent DC in the gut mucosa may be involved in an analogous process aimed at maintaining immune integrity in the face of continuous onslaught from harmless antigens. Elucidation of the characteristics and fate of mucosal DC will be important for understanding both how tolerance to food proteins and commensal bacteria is maintained and how alterations in DC function may lead to disease.

Materials and methods

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

Mice

Female BALB/c (H-2d) mice were obtained from Harlan Olac (Bicester, UK). DO11.10 mice, transgenic for TCR specific for I-Ad + OVA 323–339, were obtained originally from Dr. N. Lycke, Department of Clinical Immunology, University of Göteborg, Sweden. All mice were first used at 8 weeks of age and were maintained under SPF conditions at the University of Glasgow.

Treatment of mice with flt3 ligand

Mice were injected daily i.p. with 10 μg recombinant human flt3L (kindly provided by Amgen Inc., formerly Immunex, Seattle, WA) in saline for 7–10 days before harvest of DC. Controls received 0.2 ml saline i.p. only.

Isolation of cells from lymphoid tissues

Single-cell suspensions were prepared from MLN, spleen and peripheral lymph nodes by gently mashing through nylon mesh filters (Becton Dickinson, BD; Cowley, UK), resuspended in RPMI (Life Technologies, Paisley, UK) or by digestion for 20 min at 37°C with 100 U/ml collagenase (type VIII; Sigma, Poole, UK) and 30 μg/ml DNase I (Roche Diagnostic Ltd., Lewes, UK) in calcium- and magnesium-free Hanks’ balanced salt solution (CMF; Life Technologies) containing 20% NCS (Life Technologies). The cells were passed through Nitex mesh, counted by phase contrast microscopy and kept in CMF/20% newborn calf serum (NCS) on ice until use.

Isolation of LP and PP cells

Small intestines were washed in CMF and the PP excised. To obtain LP cells, the guts were opened longitudinally, cut into small pieces and washed thoroughly in CMF before being incubated in CMF containing 2 mM EDTA (Sigma) for 15 min in a shaker at 37°C. To remove the epithelial layer, the pieces of intestine were shaken twice thoroughly in CMF and the incubation process repeated for four cycles. The remaining fragments of intestinal tissue were then incubated with 100 U/ml collagenase and 30 μg/ml DNase I in CMF/20% NCS at 37°C for 45 min. The fragments were then disrupted by shaking and the supernatants collected. After repeating the process three times, supernatants were passed through Nitex mesh, washed and stored in CMF/20% NCS on ice until use. PP were pooled and digested with collagenase and DNase I for 30 min, washed and stored in CMF/20% NCS at 4°C until LP cell isolation had been completed.

Purification of DC from lymphoid tissues

To obtain DC, single-cell suspensions were incubated with 5 μg/107 cells biotinylated anti-CD11c antibody (clone HL3; BD PharMingen) in cold MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) in the presence of 5 μg/107 cells Fc Block (anti-CD16/32, 2.4G2; BD PharMingen) for 15 min at 4°C. After two washes, the cells were incubated with Streptavidin MicroBeads (Miltenyi Biotec, Bisley, UK) for 15 min at 4°C, washed in cold MACS buffer, filtered through Nitex mesh and positively selected using an LC MACS column (Miltenyi Biotec). Selected cells were washed once and then positively selected once more using an MS MACS column according to the manufacturer's instructions. In the case of spleen cells, two MS columns were used. Positively selected cells were routinely found to contain 80–92% CD11c+ cells by flow cytometry.

Flow cytometry

The following antibodies were used (all from BD PharMingen): PE-anti-CD11c (HL3) in combination with FITC or biotinylated antibodies against CD11b (M1/70), CD40 (3/23), CD69 (H1.2F3), CD80 (16-10A1), CD86 (GL1), MHC class II (I-Ab, 25-9-17), CD8α (53-6.7), CD4 (RM4-5), CD45R/B220 (RA3-6B2) and Ly-6C/G (RB6-8C5). Appropriate isotype-matched controls from BD PharMingen were included in all experiments. Biotinylated antibodies were detected using streptavidin conjugated to allophyocyanin (APC; BD PharMingen), and all staining procedures were carried out using 1×105 to 5×105 cells in 200 μl FACS buffer (PBS + 2% NCS and 0.05% sodium azide) for 30 min at 4°C in the dark. To exclude dead cells, 5 ng propidium iodide (PI; Sigma) was added just before analysis, and cells were analyzed using a FACSCalibur cytometer and CellQuest software.

Assessment of endocytosis by uptake of FITC-dextran

To assess endocytosis in vitro, freshly isolated cells were resuspended in RPMI/10% FCS (Harlan Sera Lab, Loughborough, UK) at 1×106 cells/ml and incubated for 5–60 min at 37°C or 0°C in 24-well ultra-low attachment polystyrene plates (UltraLow Cluster, Corning Inc., Corning, USA) with 1 mg/ml FITC-dextran (40 kDa; Sigma). The uptake was stopped by adding ice-cold PBS containing 1% FCS and 0.01% sodium azide. The cells were then washed twice in FACS Buffer and stained with PE-anti-CD11c for analysis by flow cytometry as described above. The results are given as the ΔMFI, which is the mean fluorescence intensity (MFI) obtained using live-gated (PI) CD11c+ cells incubated at 37°C minus the MFI obtained when the cells were incubated at 4°C. To assess endocytosis in vivo, mice were fed 5 mg/0.2 ml FITC-dextran, and the intestine was removed at intervals thereafter.

Assessment of APC activity in vitro

Cells to be used as APC were isolated directly from OVA-fed mice or were pulsed with 5 mg/ml OVA protein for 2 h at 37°C in 24-well ultra-low attachment polystyrene plates (1×106 cells/ml in 2 ml) before being washed twice in RPMI/10% FCS. The APC were then treated with 50 μg mitomycin c (Sigma)/107 cells/ml for 1 h, washed four times and plated in flat-bottomed 96-well plates (Costar, High Wycombe, UK) together with 2×105 OVA-specific TCR-transgenic CD4+ lymph node/spleen cells purified from DO11.10 mice using a CD4+ T cell isolation kit (Miltenyi Biotec). All cultures were performed in quadruplicate in a total volume of 200 μl RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin + 100 μg/ml streptomycin, 2 mM L-glutamine and 1.25 μg/ml Fungizone ("complete medium"; all reagents from Life Technologies) at 37°C in a humidified 5% CO2 incubator. In some experiments, 10 μg/ml OVA 323–339 was added as a positive control. Proliferation was assessed by addition of 1 μCi/well [3H]-thymidine (West of Scotland Radionucleotide Dispensary, Glasgow, UK) 18 h before harvesting and counting using a beta plate counter (Wallac, Turku, Finland).

Assessment of APC activity in vivo

Antigen-loaded APC (105) were injected s.c. into the hind footpad of BALB/c mice in 50 μl RPMI 1640 1 day after the i.v. adoptive transfer of 3×106 KJ1.26+ cells from DO11.10 mice. Before transfer, DO11.10 cells were labeled with CFSE (Molecular Probes, Oregon, USA) by incubating for 10 min at 107 cells/ml in 10 μM CFSE diluted in PBS/0.1% BSA. The clonal expansion of OVA-specific cells was determined in the draining popliteal lymph node by assessing the level of CFSE and CD69 on CD4+KJ1.26+ cells by flow cytometry. Ten days after transfer of OVA-loaded APC, mice were challenged s.c. into the opposite footpad with 10 μg OVA in 50 μl CFA (Sigma), and systemic immunity was assessed 20 days after challenge by measurement of DTH responses after footpad challenge with 100 μg heat-aggregated OVA, as described previously 36. Antigen-specific IFN-γ production was assessed in these mice by ELISA analysis of supernatants of draining lymph node cells taken 10 days after challenge with OVA/CFA. Briefly, single-cell suspensions from the lymph nodes of three mice/group were pooled and 105 cells cultured in a total volume of 200 μl in flat-bottomed tissue culture plates in complete RPMI 1640 containing 1 mg/ml OVA. Supernatants were harvested after 48 h culture and stored at –20°C before analysis of IFN-γ levels by Sandwich ELISA using paired antibodies (BD PharMingen) as described previously 36. The levels of IFN-γ were calculated using serial dilutions of recombinant IFN-γ (BD PharMingen) and expressed as ng/ml.

Assessment of cytokine mRNA expression by real-time PCR

Total RNA was prepared using TRIzol (Life Technologies) according to the manufacturer's instructions. RNA was treated with DNase I (Ambion, Austin, TX) and reverse transcribed to cDNA using Superscript reverse transcriptase (Life Technologies). cDNA levels of murine IL-12p40, IL-10, IFN-γ, type I IFN, TNF-α and HPRT were quantified by real-time PCR using an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Amplification was achieved using an initial cycle of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 50°C for 1 min. cDNA levels during the linear phase of amplification were normalized against HPRT controls. Determinations were made in triplicate and the mean ± SD determined. The forward (f), reverse (r) primers and 5′-6-carboxy-fluorescein-labeled/3′-6-carboxy-tetramethyl-rhodamine-labeled probes (p) used to detect expression of the corresponding murine genes were as follows: IL-12p40 (f 5′-GGAATTTGGTCCACTGAAATTTTAAA-3′; r 5′-CACGTGAACCGTCCGGAGTA-3′; p 5′-ACAAGACTTTCCTGAAGTGTGAAGCACCAAAT-3′); IL-10 (f 5′-ACAACATACTGCTAACCGACTCCTT-3′; r 5′-AGGTAAAACTGGATCATTTCCGATA-3′; p 5′-TGGCAACCCAAGTAACCCTTAAAGTCCTG-3′); IFN-γ (f 5′-TCAAGTGGCATAGATGTGGAAGAA-3′; r 5′-TGGCTCTGCAGGATTTTCATG-3′; p 5′-TCACCATCCTTTTGCCAGTTCCTCCAG-3′); IFN-β (f 5′-CCTACAGGGCGGACTTCAAG-3′; r 5′-GGATGGCAAAGGCAGTGTAACT-3′; p 5′-TGCATCTTCTCCGTCATCTCCATAGGGA); TNF--α (Applied Biosystems); HPRT (f 5′-GCAGTACAGCCCCAAAATGG-3′; r 5′-AACAAAGTCTGGCCTGTATCCAA-3′; p 5′-TAAGGTTGCAAGCTTGCTGGTGAAAAGGA-3′).

Immunohistochemistry

Sections of small intestine were frozen in OCT embedding medium (Bayer, Newbury, UK). Sections were cut (8 μm) and mounted on to poly-L-lysine-coated microscope slides, which were air dried and stored at –20°C until use. Sections were brought to room temperature and fixed in 4% formaldehyde in PBS for 5 min. To quench endogenous peroxidase activity, sections were incubated with three changes of 3% H2O2 and 0.1% NaN3 in PBS for 45 min before blocking with an avidin biotin blocking kit (Vector). Biotinylated anti-CD11c antibody (HL3; PharMingen) diluted in blocking reagent (Molecular Probes) was added to sections for 30 min; control sections were incubated with blocking reagent only. After washing, streptavidin-HRP (Molecular Probes), diluted in the blocking reagent, was added to all sections for 30 min. The slides were washed again, and staining was amplified and visualized using tyramide conjugated to Alexa Fluor 647 (Molecular Probes). Slides were mounted in Vectorshield containing DAPI (Vector).

Statistical analysis

Where appropriate, results expressed as means ± standard deviation were compared using Student's t-test, while Wilcoxon's Rank Sum test was used to analyze non-parametric data.

Acknowledgements

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

This work was supported by the Wellcome Trust UK, by SHERT and by the EC 5th Framework in Biotechnology. Dr. Fernando Chirdo was supported by a Research Collaboration Award from the Wellcome Trust.

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  • 3

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  • 4

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  • 5

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  • 7

    WILEY-VCH

  • 1
    Mowat, A. McI., Oral tolerance; Physiology and Clinical Implications. Curr. Opin. Gastroenterol. 1999. 15: 546556.
  • 2
    Faria, A. M. C. and Weiner, H. L., Oral tolerance: mechanisms and therapeutic applications. Adv. Immunol. 1999. 73: 153264.
  • 3
    Guermonprez, P., Valladeau, J., Zitvogel, L., Thery, C. and Amigorena, S., Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 2002. 20: 621667.
  • 4
    Viney, J. L., Mowat, A. McI., O'Malley, J. M., Williamson, E. and Fanger, N., Expanding dendritic cells in vivo enhances the induction of oral tolerance. J. Immunol. 1998. 160: 58155825.
  • 5
    Mowat, A. McI., Anatomical basis of tolerance and immunity to intestinal antigens. Nat. Rev. Immunol. 2003. 3: 331341.
  • 6
    Ruedl, C., Rieser, C., Bock, G., Wick, G. and Wolf, H., Phenotypic and functional characterization of CD11c+ dendritic cell population in mouse Peyer's patches. Eur. J. Immunol. 1996. 26: 18011806.
  • 7
    Spahn, T. W., Weiner, H. L., Rennert, P. D., Lugering, N., Fontana, A., Domschke, W. and Kucharzik, T., Mesenteric lymph nodes are critical for the induction of high-dose oral tolerance in the absence of Peyer's patches. Eur. J. Immunol. 2002. 32: 11091113.
  • 8
    Spahn, T. W., Fontana, A., Faria, A. M., Slavin, A. J., Eugster, H. P., Zhang, X., Koni, P. A., Ruddle, N. H., Flavell, R. A., Rennert, P. D. and Weiner, H. L., Induction of oral tolerance to cellular immune responses in the absence of Peyer's patches. Eur. J. Immunol. 2001. 31: 12781287.
  • 9
    Alpan, O., Rudomen, G. and Matzinger, P., The role of dendritic cells, B cells and M cells in gut-oriented immune responses. J. Immunol. 2001. 166: 48434852.
  • 10
    Fujihashi, K., Dohi, T., Rennert, P. D., Yamamoto, M., Koga, Y., Kiyono, H. and McGhee, J. R., Peyer's patches are required for oral tolerance to proteins. Proc. Natl. Acad. Sci. USA 2001. 98: 33103315.
  • 11
    Zimmer, K. P., Buning, J., Weber, P., Kaiserlian, D. and Strobel, S., Modulation of antigen trafficking to MHC class II-positive late endosomes of enterocytes. Gastroenterology 2000. 118: 128137.
  • 12
    Mayrhofer, G., Pugh, C. W. and Barclay, A. N., The distribution, ontogeny and origin in the rat of Ia-positive cells with dendritic morphology and of Ia antigen in epithelia, with special reference to the intestine. Eur. J. Immunol. 1983. 13: 112122.
  • 13
    Maric, I., Holt, P. G., Perdue, M. H. and Bienenstock, J., Class II MHC antigen (Ia)-bearing dendritic cells in the epithelium of the rat intestine. J. Immunol. 1996. 156: 14081414.
  • 14
    Iwasaki, A. and Kelsall, B. L., Freshly isolated Peyer's patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. J. Exp. Med. 1999. 190: 229239.
  • 15
    Iwasaki, A. and Kelsall, B. L., Unique functions of CD11b+, CD8α+ and double negative Peyer's patch dendritic cells. J. Immunol. 2001. 166: 48844890.
  • 16
    Huleatt, J. W. and Lefrancois, L., Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8+ T cells in vivo. J. Immunol. 1995. 154: 56845693.
  • 17
    Harper, H., Cochrane, L. and Williams, N. A., The role of small intestinal antigen-presenting cells in the induction of T cell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance. Immunology 1996. 89: 449456.
  • 18
    Becker, C., Wirtz, S., Blessing, M., Pirhonen, J., Strand, D., Bechthold, O., Frick, J., Galle, P. R., Autenrieth, I. and Neurath, M. F., Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells. J. Clin. Invest. 2003. 112: 693706.
  • 19
    Wakkach, A., Fournier, N., Brun, V., Breittmayer, J. P., Cottrez, F. and Groux, H., Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 2003. 18: 605617.
  • 20
    O'Keeffe, M., Hochrein, H., Vremec, D., Caminschi, I., Miller, J. L., Anders, E. M., Wu, L., Lahoud, M. H., Henri, S., Scott, B., Hertzog, P., Tatarczuch, L. and Shortman, K., Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8(+) dendritic cells only after microbial stimulus. J. Exp. Med. 2002. 196: 13071319.
  • 21
    Kelsall, B. L. and Rescigno, M., Mucosal dendritic cells in immunity and inflammation. Nat. Immunol. 2004. 5: 10911095.
  • 22
    Bilsborough, J., George, T. C., Norment, A. and Viney, J. L., Mucosal CD8α+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties. Immunology 2003. 108: 481492.
  • 23
    Bell, S. J., Rigby, R., English, N., Mann, S. D., Knight, S. C., Kamm, M. A. and Stagg, A. J., Migration and maturation of human colonic dendritic cells. J. Immunol. 2001. 166: 49584967.
  • 24
    Martin, P., Del Hoyo, G. M., Anjuere, F., Arias, C. F., Vargas, H. H., Fernandez, L. A., Parrillas, V. and Ardavin, C., Characterization of a new subpopulation of mouse CD8α+ B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential. Blood 2002. 100: 383390.
  • 25
    Groux, H., O'Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E. and Roncarlo, M. G., A CD4+ T cell subset inhibits antigen-specific T cell responses and prevents colitis. Nature 1997. 389: 737742.
  • 26
    Moser, M., Dendritic cells in immunity and tolerance-do they display opposite functions? Immunity 2003. 19: 58.
  • 27
    Williamson, E., Westrich, G. M. and Viney, J. L., Modulating dendritic cells to optimize mucosal immunization protocols. J. Immunol. 1999. 163: 36683675.
  • 28
    Williamson, E., Bilsborough, J. M. and Viney, J. L., Regulation of mucosal dendritic cell function by receptor activator of NF-κ B (RANK)/RANK ligand interactions: impact on tolerance induction. J. Immunol. 2002. 169: 36063612.
  • 29
    Alpan, O., Bachelder, E., Isil, E., Arnheiter, H. and Matzinger, P., ‘Educated’ dendritic cells act as messengers from memory to naive T helper cells. Nat. Immunol. 2004. 5: 615622.
  • 30
    Bilsborough, J. and Viney, J. L., Gastrointestinal dendritic cells play a role in immunity, tolerance, and disease. Gastroenterology 2004. 127: 300309.
  • 31
    Akbari, O., DeKruyff, R. H. and Umetsu, D. T., Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat. Immunol. 2001. 2: 725731.
  • 32
    Liu, L. and MacPherson, G., Antigen acquisition by dendritic cells: intestinal dendritic cells acquire antigen administered orally and can prime naive T cells in vivo. J. Exp. Med. 1993. 177: 12991307.
  • 33
    Scheinecker, C., McHugh, R., Shevach, E. M. and Germain, R. N., Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. J. Exp. Med. 2002. 196: 10791090.
  • 34
    Wilson, N. S., El-Sukkari, D., Belz, G. T., Smith, C. M., Steptoe, R. J., Heath, W. R., Shortman, K. and Villadangos, J. A., Most lymphoid organ dendritic cell types are phenotypically and functionally immature. Blood 2003. 103: 21872195.
  • 35
    Carbone, F. R., Belz, G. T. and Heath, W. R., Transfer of antigen between migrating and lymph node-resident DCs in peripheral T cell tolerance and immunity. Trends Immunol. 2004. 25: 655658.
  • 36
    Garside, P., Steel, M., Worthey, E. A., Satoskar, A., Alexander, J., Bluethmann, H., Liew, F. Y. and Mowat, A. McI., Th2 cells are subject to high dose oral tolerance and are not essential for its induction. J. Immunol. 1995. 154: 56495655.