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Keywords:

  • Cell migration;
  • Hypersensitivity;
  • In vivo microscopy;
  • Regulatory T cells

Abstract

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

CD4+CD25+ regulatory T cells (Treg) exert suppressive functions on effector T cells in vitro and in vivo. However, the exact cellular events that mediate this inhibitory action remain largely unclear. To elucidate these events, we used intravital microscopy in a model of contact hypersensitivity (CHS) and visualized the leukocyte-endothelium interaction at the site of antigen challenge in awake C57BL/6 mice. Injection of Treg i.v. into sensitized mice at the time of local hapten challenge significantly inhibited rolling and adhesion of endogenous leukocytes to the endothelium. A similar inhibition of leukocyte recruitment could be recorded after injection of Treg-derived tissue culture supernatant. Thus, these data indicate that soluble factors may account for the suppressive effects. Accordingly we found that IL-10, but not TGF-β, was produced by Treg upon stimulation and that addition of anti-IL-10 antibodies abrogated the suppressive effects of Treg and tissue culture supernatant in CHS reactions. Moreover, CD4+CD25+ T cells isolated from IL-10–/– mice were not able to suppress the immune response induced by hapten treatment in C57BL/6 mice. In conclusion, our data suggest that cytokine-dependent rather than cell-cell contact-dependent mechanisms play a pivotal role in the suppression of CHS reactions by Treg in vivo.

Abbreviations:
CHS:

contact hypersensitivity

dLN:

draining LN

LC:

Langerhans’ cell

rm:

recombinant mouse

TNCB:

2,4,6-trinitro-1-chloro-benzene

Introduction

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

Contact hypersensitivity (CHS) accounts for one of the most common inflammatory dermatoses in industrial countries 1. It is readily reproduced in mice and serves as an excellent model of T cell-mediated inflammation. The extent of ear swelling in hapten re-exposed sensitized mice enables the study of cellular mechanisms and assessment of the efficacy of therapeutic interventions. During sensitization, the hapten permeates the skin and binds covalently to any cell-associated or extracellular protein, forming a hapten-carrier complex that is then taken up by local dendritic cells, the Langerhans’ cells (LC). After antigen uptake, LC leave the epidermis and migrate into the dermis, where they travel via afferent lymphatic into the T cell-rich areas of the regional LN to present antigen-MHC complexes to T cells. Following a second contact with the same antigen, the LC again present the hapten-protein complex to antigen-specific memory T lymphocytes that infiltrate the antigen-exposed tissue, inducing an inflammatory reaction by recruiting more inflammatory cells to the hapten-treated area and by releasing inflammatory cytokines. While CD8+ T cells mediate initiation of the CHS immune response, CD4+ T cells are operative during its down-regulation phase 24.

Among CD4+ T cells, many subpopulations can be distinguished by their cytokine profile and/or surface molecule expression. Recently, a CD4+CD25+ subset has been identified that is characterized by its ability to suppress T cell proliferation in vivo and in vitro. These "naturally occurring" CD4+CD25+ regulatory T cells (Treg), which express the α chain of the IL-2 receptor, originate from the thymus 5, 6 and represent 5–10% of the adult peripheral CD4+ T cells in normal naive mice. It is well established that Treg participate in the maintenance of immunological self-tolerance and the regulation of immune responses to non-self antigens 7. In vitro, Treg are naturally anergic to TCR stimulation; they do not secrete IL-2 and have the capacity to inhibit the activation/proliferation of other T cells independent of cytokines 8. They have proven to be potent suppressors in a number of in vivo disease models including gastritis, thyroiditis, inflammatory bowel disease and insulin-dependent diabetes 912. Yet, many questions remain unanswered regarding the mechanisms underlying the suppressive capacity of Treg in vivo.

Here we set out to investigate the effects of natural Treg on CHS reactions and to analyze the mechanisms responsible for their suppressive function(s) in our models. We show that Treg are potent suppressors of hapten-induced contact allergy without immigrating to the affected tissues. Using a novel model of a skinfold chamber combined with in vivo microscopy, we were able to show that Treg block the adherence and extravasation of effector T cells into the "challenged" tissues. Likewise, tissue culture supernatant derived from cultured Treg and the cells themselves were equally potent "suppressors", suggesting that soluble factors are responsible for the suppressive effects. Moreover, we could demonstrate that Treg isolated from IL-10-deficient mice fail to suppress the hapten-induced immune reaction. Thus, these data indicate that the suppression of allergic contact dermatitis by CD4+CD25+ Treg is not necessarily associated with cell-cell contact; instead IL-10 is assumedly involved in the regulatory function of natural Treg in CHS reactions.

Results

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

Treg suppress the ear-swelling response in CHS reaction

First we examined the in vivo capability of naive Treg to suppress the ear-swelling reaction upon challenge in the CHS model. Treg (5 × 106 cells) isolated and pooled from LN and spleens of naive mice were injected i.v. into epicutaneously 2,4,6-trinitro-1-chloro-benzene (TNCB)-sensitized BALB/c mice immediately prior to TNCB challenge on one ear. The immune response was determined by measuring the ear-swelling reaction 24 h after injection. Injection of Treg caused a significant reduction of the ear-swelling response (Fig. 1A). In contrast, application of non-regulatory CD4+CD25 T cells had no inhibitory effect on the CHS reaction as compared to control mice that were not treated with cells. The suppressive effect of Treg was not diminished over the following 5 days (Fig. 1B), demonstrating that it is not a transient effect of the Treg. To further investigate whether Treg suppress in an organ-specific manner, Treg derived from different lymphoid organs were isolated separately and injected. Regardless of the source of cells, there was clear suppression of the CHS response (Fig. 1C). Therefore, we conclude that the suppressive function of Treg on the CHS immune response is independent of their lymphoid organ of origin.

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Figure 1. Suppression of CHS reaction upon injection of Treg. BALB/c mice were sensitized with TNCB. Five days later Treg or conventional T cells were injected followed by application of TNCB to one ear. Control mice were not treated with cells. Ear-swelling response was measured 24 h to 6 days later. (A) Treg pooled from LN and spleen significantly suppressed the ear-swelling reaction. Data shown represent the mean of the absolute increase of the ear thickness ± SD of five independent experiments, each with five mice per group (*p<0.05). (B) Treg-mediated suppression is not a transient effect. Shown are the results from four different control mice and four mice treated with Treg. (C) Treg reduce the CHS immune reaction independent of the source of isolation (LN or spleen).

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To determine the migration pattern and thus the site of activity of the injected cells, we labeled the isolated Treg and the conventional CD4+CD25 T cells with the fluorescent dye PKH26-PE before injection. The time course of migration (Fig. 2B) shows that 15 min after injection, Treg had already migrated into the draining LN (dLN) and spleen. The dye-labeled Treg represented 2.5% of CD4+ cells in the spleen and 1.3% in the draining auricular LN 24 h after challenge (Fig. 2A). Conventional T cells, which had no suppressive effects on the ear-swelling reaction, were likewise located in both the spleen and dLN (2.6% of CD4+ spleen cells and 1.2% of CD4+ LN cells). The migration pattern of both cell subpopulations was the same in control mice, which were only sensitized.

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Figure 2. Migration pattern of injected T cells. TNCB-sensitized BALB/c mice were i.v. injected with PKH26-PE-labeled Treg or respective controls directly before challenge. One control group was not challenged. (A) After 24 h FACS analysis of dLN, spleen and epidermal (ear) cells was performed. The Treg as well as the non-regulatory T cells migrated to spleen and dLN to the same extent. Treg in unchallenged control mice showed the same homing. Shown are CD4+ T cells gated out of the lymphocyte population. The dot plots for the ear epidermal cells show the whole lymphocyte population. One typical experiment out of five is shown (n.d., not detectable). (B) Time course of Treg migration in sensitized and challenged mice. One typical experiment out of two is shown.

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To further test whether injected Treg also home to the site of inflammation, cryosections of TNCB-challenged ears from the different experimental groups were analyzed by immunofluorescence microscopy. No infiltration by PKH26-PE-labeled Treg or non-regulatory T cells could be detected (data not shown). We also analyzed epidermal cells isolated from the ears of the differently treated mice by FACS to examine possible immigration of fluorescence-labeled Treg at different time points (15 min, 30 min, 1 h, 4 h, 24 h and 48 h) after injection. As in the cryosections, no PKH-PE-labeled Treg could be detected at any time point (Fig. 2A, 24 h after challenge). These results indicate that no Treg, or only a non-detectable fraction, migrate into the site of inflammation.

Treg prevent hapten-induced leukocyte-endothelium interactions

Despite their absence at the site of inflammation, injected Treg significantly reduced the inflammatory ear-swelling reaction. Therefore, we set out to directly visualize the migration pattern of injected Treg to identify their site of action during the CHS reaction. For that purpose, we used intravital microscopy in the dorsal skinfold chamber preparation in awake mice (Fig. 3A). Application of hapten to the skin within the observation chamber enabled direct visualization of the leukocyte-endothelium interaction during CHS and hence quantification of the potential inhibitory effect of Treg application. In general, 5 days after epicutaneous sensitization with TNCB, CD4+CD25 cells or Treg were injected i.v. immediately before challenge with TNCB (or carrier substance) at the skinfold chamber. Thereafter, the fluorescence marker Rhodamine 6G, which stains all leukocytes, was injected. Using intravital microscopy we were able to detect the rolling and sticking of Rhodamine-labeled leukocytes to endothelial cells at the site of hapten application (Fig. 3B and supplemental movie data).

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Figure 3. Treg prevent leukocyte-endothelium interactions. Two titanium frames were implanted on the entire back of C57BL/6 mice. In a circular area, one layer of the skin was completely removed. The remaining layer was covered with a glass coverslip incorporated into one of the frames (A). Mice were sensitized with 1% TNCB on the same day the implantation of the skinfold chamber was carried out. After 5 days, mice were injected with CD4+CD25 or CD4+CD25+ T cells and Rhodamine immediately before challenging. Time courses of numbers of slow-rolling (C) and adhesive (D) leukocytes were measured by intravital fluorescence microscopy (B; arrows point out individual cells). Leukocyte-endothelium interactions increased in the control group (n=5) and in mice injected with CD4+CD25 T cells (n=10). Injection of Treg (n=10) reduced the leukocyte-endothelium interaction to baseline levels from the beginning. Shown are means of n mice ± SEM (significance control group vs. acetone/oil-treated group, +p<0.05; group injected CD4+CD25 T cells vs. mice injected Treg, *p<0.05).

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Under baseline conditions (acetone/oil), the majority of circulating leukocytes did not interact with the endothelium, and the flow of Rhodamine-positive cells was unchanged compared to non-treated mice (supplemental movie 1). Challenge with hapten caused a significant increase in slow rolling and adhesion of leukocytes during an observation period of 4 h (Fig. 3C, D and supplemental movie 2). In this newly established CHS model, the peak of inflammation is reached within 4 h after antigen challenge and lasts for ∼12 h. Thereafter, the down-regulation phase of the CHS reaction takes place.

Next we investigated the effects of Treg or control CD4+CD25 T cells on the leukocyte-endothelium interaction. Analysis of several experiments revealed that injection of Treg reduced the leukocyte-endothelium interaction to the baseline levels measured in control mice challenged with solvent only (Fig. 3C, D and supplemental movie 3); 30 min after injection of the Treg, we could detect fewer rolling and sticking leukocytes as compared to control mice treated with hapten only. In contrast, application of CD4+CD25 T cells did not influence the leukocyte-endothelium interaction in hapten-treated mice.

To further define whether injected Treg migrate into the inflamed tissue, Treg as well as control T cells were stained with the fluorescent dye PKH-FITC before injection and were traced by immunofluorescence microscopy. Influx of circulating effector T cells into the site of inflammation was significantly suppressed after injection of syngeneic Treg. However, at the same time, we could not identify any PKH-FITC-labeled cells, indicating the absence of injected Treg in the observed vessels. Moreover, in mice implanted with the skinfold chamber, Treg migrated into the dLN and the spleen within 30 min after injection, thus show a homing pattern comparable to the CHS model (data not shown). Thus, these results show that Treg indeed prevent influx of circulating effector T cells into inflamed tissue but do not migrate to the site of inflammation themselves.

To analyze CD8+ effector T cells in the inflamed tissue, we performed FACS analysis of isolated epidermal cells derived from challenged ears. Here we detected reduced influx of CD8+ T cells during CHS reaction in the mice treated with Treg (Table 1). In addition, tissue sections of mouse ears were obtained and stained with hematoxylin/eosin for morphological evaluation or with anti-CD8 antibodies. Fig. 4A displays histological images of ear sections 24 h after challenge, illustrating the increased ear edema following application of hapten. Treatment with Treg reduced the edema (Fig. 4A), and immunofluorescence revealed a decrease in infiltrating CD8+ T cells (Fig. 4B). Moreover, in the skinfold chamber model using non-sensitized mice, which did not harbor antigen-specific CD8+ T cells, hapten challenge caused only a slight increase in leukocyte-endothelium interaction comparable to the response measured in sensitized mice challenged with solvent only (supplemental movie 4). In both models, injection of Treg showed no measurable effect on the results. These data verify that the suppressive effect of injected Treg during CHS is mediated by the inhibition of CD8+ effector T cells directly or indirectly.

Table 1. Injection of Treg reduces the influx of CD8+ effector T cells into the site of inflammation
Experimental groupsa)

Epidermal

CD8+ T cells [% ± SD]b)

  1. a) Each experimental group consisted of three mice. Mice were sensitized on day 0, and treatment with Treg (CD4+CD25+) or conventional T cells (CD4+CD25) occurred on day 5, 10–15 min before challenge with hapten (TNCB) or solvent (acetone/oil) only.

  2. b) Leukocytes were isolated from the epidermis of the challenged ears 24 h after challenge and analyzed by FACS. Data show the percentage of CD8+ T cells in the epidermal leukocyte population as the mean of three experiments ± SD.

  3. c) Injection of Treg significantly reduced the influx of CD8+ T cells into the site of inflammation (*p<0.05).

 SensitizationTreatmentChallenge 
1TNCB___TNCB4.32±2.24
2TNCB___acetone/oil0.40±0.24
3TNCBCD4+CD25+ i.v.c)TNCB1.58±0.59*c)
4TNCBCD4+CD25 i.v.TNCB4.19±2.17
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Figure 4. Treg reduce edema and the infiltration of CD8+ T cells. Tissue sections from mouse ears were obtained 24 h after challenge with hapten or solvent and stained with hematoxylin/eosin or fluorochrome-conjugated anti-CD8 antibodies. (A) Treatment with Treg (CD4+CD25+) reduced edema following application of hapten compared to the mice that received conventional T cells (CD4+CD25). "Baseline" demonstrates ear swelling after application of solvent only. The blue dots, exemplified by the black arrows, represent leukocytes infiltrating the ear tissue. (B) Infiltration of CD8+ effector T cells into the hapten-treated ear, as a result of the immune reaction, was decreased after injection of Treg. White arrows point at some examples of FITC-stained CD8+ T cells in the ear sections.

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Soluble factors produced by cultured Treg inhibit the CHS reaction

Since we could not detect any Treg in the inflamed tissue, we reasoned that soluble factors rather than cell-cell contact are operative in the suppression of the CHS reaction. Accordingly, isolated Treg and CD4+CD25 T cells were cultured in the presence of anti-CD3 and anti-CD28 antibodies. Subsequently, supernatants were harvested and injected i.v. into mice prior to challenge with TNCB. As shown in Fig. 5A, injection of supernatant derived from activated Treg caused a significant, dose-dependent suppression of the inflammatory reaction. In contrast, injection of supernatant from conventional CD4+CD25 T cells or medium alone had no effect on the CHS reaction as compared to control mice. Similar results were obtained in experiments using the skinfold chamber model in C57BL/6 mice. Here, the leukocyte-endothelium interaction within the hapten-exposed tissue was reduced to baseline levels by injection of 100 μL supernatant from stimulated Treg (Fig. 5B, C). This effect remained evident over the entire observation period. Consistent with the inability of injected CD4+CD25 T cells to suppress the TNCB-induced inflammatory reaction, supernatant from activated CD4+CD25 T cells also had no inhibitory effect on the leukocyte-endothelium interaction. According to these results, the suppressive function of the isolated Treg in allergic contact dermatitis is mainly contact-independent and is at least in part mediated by soluble factors produced by activated Treg.

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Figure 5. Supernatant produced by cultured Treg suppresses the CHS response significantly, associated with the inhibition of leukocyte-endothelium interactions. Treg or conventional T cells were cultured for 48 h, and supernatants (SN) were harvested and injected immediately before challenge. Ear-swelling reaction was measured 24 h later. (A) Supernatant produced by Treg suppressed the ear-swelling response in a dose-dependent manner. Supernatants of CD4+CD25 T cells or medium alone had no influence on the immune response. Data shown represent the mean of the absolute increase in ear thickness ± SD of three independent experiments, each with five mice per experimental group (*p<0.05). (B, C) C57BL/6 mice were sensitized, and skinfold chambers were implanted. Five days later 100 μL supernatant produced by 48 h-cultured T cells or freshly isolated CD25+ or CD25- T cells were injected, followed by challenging and injection of Rhodamine. Soluble factors produced by Treg (n=8) suppressed the leukocyte-endothelium reaction for 4 h. In contrast, supernatant of conventional T cells (n=6) had no effect on rolling (B) or adherence (C) of leukocytes. Shown are means of n mice ± SEM (*p<0.05, +p<0.05).

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Supernatant of activated Treg contains high amounts of IL-10

To identify soluble factors produced by Treg that may be responsible for the suppressive effect on CHS reactions, we quantified the anti-inflammatory cytokines IL-10 and TGF-β in the tissue culture supernatants of stimulated or non-stimulated T cells by ELISA. The amount of IL-10 was significantly increased in the supernatant of stimulated Treg (1711±159 pg/mL) as compared to non-regulatory T cells (311±86 pg/mL) (Fig. 6A). Unstimulated T cells produced virtually no IL-10 under these conditions (CD4+CD25 7±2 pg/mL, CD4+CD25+ 36±6 pg/mL). To prove the specificity of this assay, neutralizing anti-IL-10 antibodies were added to some samples, which resulted in the complete neutralization of IL-10 in the cultures. We could not detect substantially increased amounts of TGF-β in the tissue culture supernatants of Treg (Fig. 6B). However, only trace amounts of TGF-β were detected in any T cell cultures.

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Figure 6. Supernatant of activated Treg contains high amounts of IL-10. Isolated cells were cultured for 48 h ± anti-CD3 and anti-CD28. Thereafter, levels of IL-10 (A) and TGF-β (B) in the supernatants (SN) were determined by ELISA. Only IL-10 was detectable in the supernatants produced by stimulated (stim) CD4+CD25 and especially by stimulated Treg (A). Data shown represent the mean ± SD of five independent experiments (*p<0.05). (C) A volume of 20 μL of the indicated supernatant was added into the culture of freshly isolated and stimulated CD4+CD25 T cells. Supernatant produced by activated Treg significantly suppressed proliferation. Neutralization of IL-10 reversed this inhibition completely, whereas anti-TGF-β did not influence the suppressive function of Treg in vitro. Data shown represent the mean ± SD of three independent experiments (*p<0.05).

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To further demonstrate the impact of Treg-derived IL-10 on T cell proliferation, supernatants were added to freshly isolated and stimulated CD4+CD25 T cells in vitro. These experiments (Fig. 6C) show that tissue culture supernatants from stimulated Treg significantly suppressed the proliferation of conventional CD4+ T cells (by ∼60%). In accordance with our previous findings, the addition of neutralizing antibodies against IL-10 blocked the suppressive effects, whereas addition of anti-TGF-β had no effect on the inhibitory activity. Supernatants obtained from CD4+CD25 T cells did not effect the proliferation of CD4+ T cells. Thus, these data indicate that IL-10 plays a major role in the regulatory function of Treg.

IL-10 is required for the regulatory function of Treg in vivo

To analyze the role of IL-10 and/or TGF-β in the Treg-mediated suppression of CHS reactions, mice were sensitized with TNCB, and 5 days thereafter anti-IL-10 or anti-TGF-β antibodies were injected before application of 100 μL Treg-derived tissue culture supernatant (corresponding to ∼171 pg IL-10, as determined by ELISA). Fig. 7A shows that injection of anti-IL-10 antibodies completely blocked the suppressive effect of Treg-derived supernatant. Neutralization of TGF-β had no influence on the suppressive capacity of Treg supernatant (Fig. 7B). However, administration of up to 1250 pg recombinant mouse (rm)IL-10 instead of Treg-derived supernatant or the cells themselves did not suppress the ear-swelling reaction following hapten challenge (data not shown). The same results were obtained in experiments with the dorsal skinfold chamber. Administration of 750 pg rmIL-10 did not affect the leukocyte-endothelium interaction during inflammatory reactions (Fig. 7C). Thus, to further analyze the importance of IL-10 in Treg-mediated suppression, we injected Treg isolated from IL-10-deficient mice (IL-10–/– mice) into sensitized wild-type mice before challenge. Fig. 7D shows that Treg from IL-10–/– mice failed to inhibit the CHS reaction, while showing the same migration pattern as wild-type Treg (data not shown), indicating that production of IL-10 is essential for the Treg to mediate their immunosuppressive function in vivo.

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Figure 7. IL-10 is required for the suppressive function of Treg in vivo. BALB/c mice were sensitized, and 5 days later anti-IL-10 (A) or anti-TGF-β (B) was administrated by i.p. injection, 100 μL supernatant (SN) from 48 h-cultured cells was injected i.v. and challenge was performed. The ear-swelling response was measured 24 h later. (A) Suppression of the immune response by soluble factors produced by Treg was completely reversed after neutralization of IL-10. (B) Anti-TGF-β did not affect the suppressive function. Data shown represent the mean of the absolute increase in ear thickness ± SD of three independent experiments, each with five mice per experimental group (*p<0.05). (C) rmIL-10 was not sufficient to reduce the number of rollers. Shown are means of five mice ± SEM (+p<0.05). (D) Treg isolated from IL-10-deficient mice failed to suppress the ear-swelling reaction caused by hapten treatment. Data shown represent the mean of the absolute increase of the ear thickness ± SD of four independent experiments, each with five mice per experimental group (*p<0.05).

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Discussion

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

In our study we analyzed the regulatory function of adoptively transferred Treg in a murine model of allergic contact dermatitis and demonstrated the inhibitory potential of Treg in CHS responses. These data are in accordance with recent observations by Dubois et al., who described that in oral tolerance Treg abrogate the development of hapten-specific CD8+ T cells in vivo, averting CHS reactions 13, 14.

Using the skinfold chamber, we could show for the first time that suppression of the CHS reaction by Treg is associated with a reduction in the influx of endogenous leukocytes and with an inhibition of leukocyte-endothelium interactions directly at the site of inflammation. This suppressive activity affects antigen-specific CD8+ leukocytes, which mediate the inflammatory response in CHS reactions 15. Accordingly, the number of CD8+ T cells was significantly diminished in the hapten-induced ear reaction after application of Treg in our studies.

These effects were detectable within few minutes after injection, and as Treg were not present at the site of inflammation, we conclude that cell-cell contact in the affected tissues is most likely not required to exert the inhibitory functions in CHS reactions.

The rather rapid hapten-induced interaction of leukocytes with endothelial cells observed in our study is in accordance with findings of Hwang and coworkers, who described a first peak of leukocyte recruitment 2 h after challenge with a hapten. The final magnitude of the inflammatory reaction is further correlated with the efficiency of leukocyte recruitment in the early phase 16, and thus, rapid blockade of leukocyte adherence by Treg is crucial for efficient reduction of the CHS response.

How the injected Treg mediate their suppressive activity is still elusive. Numerous data support a requirement for cell-cell contact. However, these experiments were mainly done in vitro, whereas several in vivo studies suggest that other (soluble) factors may play essential roles. For example, in murine models of inflammatory bowel diseases, symptoms could only be prevented by transfer of IL-10-producing CD4+CD25+ T cells, as administration of antibodies against IL-10 or its receptor neutralized the suppressive effects of the Treg. Moreover, transfer of Treg from IL-10-deficient mice failed to prevent colitis 17, 18. In other colitis models, the injection of freshly isolated or activated Treg halted the progression of the disease and reversed the symptoms. These effects were critically dependent on IL-10, TGF-β and CTLA-4, since injection of the mice with anti-IL-10 receptor, anti-CTLA-4 or anti-TGF-β abrogated the therapeutic effects of the transferred Treg 19. Even in a model of UV-induced Treg, IL-10 seems to be crucial, since injection of anti-IL-10 immediately after cell transfer prevented the suppression of the CHS reaction 20. IL-10, produced by dLN CD4+ T cells, is also required in a murine low-zone tolerance model using contact allergens, where it is crucial for the generation of CD8+ regulatory cells 21. In line with these observations, we demonstrated that the supernatant of Treg was as potent as the cells themselves in suppressing the inflammatory response in the CHS model. Administration of antibodies to IL-10 reversed the suppression completely, whereas neutralization of TGF-β was without any detectable effect on the ear-swelling reaction. Moreover, Treg isolated from IL-10-deficient mice failed to suppress the CHS immune response. Thus, we conclude that IL-10 is required for the Treg-induced regulation of murine CHS.

Possible means as to how IL-10 accomplishes the inhibition of adherence can be deduced from other reports showing that IL-10 is able to reduce the monocyte/endothelium interaction by reducing the induction of endothelial E-selectin by monocytes 22. Another possible mechanism is IL-10-induced down-regulation of TNF-α at the site of inflammation, which subsequently results in abrogation of the up-regulation of endothelial ICAM--1 and inhibition of CHS, as shown for IL-10 produced by UV-induced suppressor T cells 23. A similar mechanism was described by Sasaki et al., who demonstrated that in IL-10-transfected endothelial cultures, the expression of TNF-α-induced MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is significantly reduced and, consequently, the MAdCAM-1-dependent lymphocyte adhesion inhibited 24. Therefore, one can speculate that in our model, IL-10, either directly secreted by Treg or induced by the administration of exogenous Treg, down-modulates adhesion molecules on the affected endothelium and thus prevent extravasation of leukocytes.

However, this requirement does not imply that IL-10 acts directly as an effector of the observed suppression. In fact, it is possible that IL-10 acts as a "sensitizing" agent by priming the Treg for their suppressive function. Moreover, the local site of production and/or secretion as well as the time frame seem to be important, since injection of rmIL-10 does not necessarily mimic the effects of Treg injection. In our experiments we were not able to suppress the elicitation phase of the CHS reaction by rmIL-10 administration; this was also shown by Schwarz et al., who demonstrated that effects of rmIL-10 on the elicitation phase of CHS reactions could only be observed when IL-10 was administered i.p. at least 12 h before hapten challenge 25 and by Kondo et al., who had to inject the rmIL-10 intradermally into the site of antigen challenge directly before challenge 26.

These apparent differences in function between injected rmIL-10 and Treg-derived IL-10 may be explained by the tissue environment, i.e. injected Treg secrete IL-10 into their local environment and thus cells in their vicinity will be affected directly, whereas i.v. injection of IL-10 may not reach the crucial T cell populations due to proteolytic decay, dilution and physical barriers. However, other factor(s) that, in addition to IL-10, are necessary to drive the suppressive capacity of the Treg could be involved. One potential candidate would be the membrane-bound form of TGF-β, which is discussed as a possible marker for Treg 27. Treg expressing TGF-β may suppress effector CD8+ T cells either directly through TGF-β/TGF-β receptor interaction or indirectly by blocking antigen-presenting cells, which would underline the necessity of cell-cell contact in addition to the soluble factor IL-10.

Evidence that the spatial distribution of Treg further affects their function derives from reports showing that UV-induced Treg do not migrate into the skin after i.v. injection but are able to suppress induction of a CHS reaction 20. In this experimental setup, injection of UV-induced Treg suppressed the sensitization phase in the LN, whereas elicitation was not affected. On the other hand, the very same UV-induced Treg blocked elicitation of the CHS response after direct injection into the site of hapten challenge (the ear). Therefore, one can speculate that only a few Treg that eventually enter the LN are potent enough to prevent sensitization of an animal, but this does not affect already activated effector T cells at the site of inflammation and thus does not block elicitation of the CHS reaction in primed animals. However in both CHS models we used, i.v. injected Treg showed homing patterns similar to UV-induced Treg, i.e. they migrated to LN and to spleen. Yet, contrary to the previous report on UV-induced Treg, in our studies the naturally occurring Treg exerted inhibitory functions during the elicitation phase of CHS.

In conclusion, we have demonstrated that IL-10 produced by CD4+CD25+ Treg is involved in suppression of CD8+ T cell-mediated immune responses in the skin after exposure to contact allergens. The mechanisms that are responsible for the induction of IL-10 production and further factors that may be responsible for the activation of Treg after challenge with allergens have to be identified in order to find new therapies for autoimmune diseases.

Materials and methods

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

Mice

BALB/c and C57BL/6 mice were purchased from Charles River (Sulzfeld, Germany) and bred at the central animal facility of the University of Heidelberg. IL-10–/– mice on a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were used for experiments at the age of 6–8 wks, and within a single experiment, mice were sex- and aged-matched.

Purification of T cell subsets

T cell subsets were always isolated from peripheral LN and spleen of naive mice. They were either used separately or pooled before injection. CD4+ T cell subpopulations were isolated using magnetic bead based separation (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+ cells were negatively selected (average purity >97%). CD4+ T cells were incubated with anti-CD25-PE mAb and then anti-PE Micro Beads. The CD4+CD25+ T cell subpopulation was positively selected according to the manufacturer's instructions (Miltenyi Biotec). The regulatory function of the Treg was always tested in proliferation assays in coculture with CD4+CD25 T cells stimulated with anti-CD3 and anti-CD28 (BD Biosciences, Heidelberg, Germany).

Fluorescence labeling

Freshly isolated or stimulated CD4+CD25 T cells and CD4+CD25+ Treg were labeled with the membrane inserting dyes PKH2 or PKH26 (Sigma-Aldrich, Taufkirchen, Germany). Serum-free cells (2 × 107) were resuspended in 1 mL diluent A (PKH2) or 1 mL diluent C (PKH26) before adding 1 mL freshly prepared membrane linkers (4 μM in the corresponding diluent). After 3 min incubation at room temperature, the reaction was stopped with 2 mL FCS within 1 min. Medium (4 mL) was added, and labeled cells were collected by centrifugation (400 × g, 10 min, 25°C). During the staining procedure, about 20% of inserted cells were lost. Between 90 and 95% of the fluorescently labeled cells were viable as assessed by Trypan blue staining. Dye insertion did not influence proliferation of or cytokine production by these cells, as determined by in vitro assays.

Contact hypersensitivity (CHS)

For the CHS reaction on mouse ears, mice were sensitized by application of 15 μL 1% TNCB (Sigma-Aldrich) dissolved in acetone/oil (4:1) on the shaved abdomen on day 0. On day 5, ear thickness was measured using a mechanical length-measuring instrument (Oditest, Kroeplin, Schlüchtern, Germany). Afterwards mice were challenged by application of 10 μL 0.5% TNCB solution dissolved in acetone and olive oil (4:1) on each side of the right ear. After 24 h the ear thickness was measured again, and the difference was calculated as Δ ear response in mm × 10–2 (mean ± SD).

For CHS in the skinfold chamber model, mice were sensitized on day 0, as described above. For intravital microscopy, we used the dorsal skinfold chamber preparation, as described previously 28. Briefly, two titanium frames were implanted into the dorsal skinfold of anesthetized mice. In a circular area, one layer of the skin was completely removed, and the remaining layer was covered with a cover slip incorporated in one of the frames. To visualize endogenous leukocytes, mice were injected with Rhodamine 6G (Sigma-Aldrich). When mice had recovered from anesthesic intravital microscopic images were recorded with the high-resolution black and white camera Spot RT KE Monochrome w/o IR Filter (Visitron, Munich, Germany) and analyzed with SpotTM software. Sequences of 1 min were recorded in 1–2 microvessel segments per chamber at baseline (prior to antigen re-exposure) and 30 min, 60 min, 120 min and 240 min after challenge with 10 µL 0.1% TNCB dissolved in acetone/oil (1:4) on the skin on the back site of the observation window.

Preparation of epidermal cell suspensions and immunohistochemistry of ear sections

Epidermal cells were obtained from mouse ear halves by trypsinization for 30 min at 37°C. Isolated cells were analyzed by flow cytometry. Thin tissue sections from mouse ears of the different experimental groups were saturated with 20% sucrose, mounted in Tissue-Tek mounting-freezing media (Miles, Torrance, CA) and sectioned using a cryostat (Leica). Sections were either stained with hematoxylin/eosin for morphological evaluation or with fluorochrome-conjugated anti-CD8 antibody (Caltag Laboratories, Burlingame, CA) for immunofluorescence analysis.

Treatment with cells or supernatant and neutralization of cytokines

Isolated and, if required, PKH-labeled CD4+CD25+ or CD4+CD25 T cells were injected i.v. immediately before challenge. In all experiments using isolated cells, we administered 5 × 106 cells per mouse.

Supernatant was produced by incubation of the isolated cells in complete medium ± anti-CD3 and anti-CD28 for 48 h. The cells (2 × 106 cells/mL medium) were pipetted into 96-well flat-bottom plates, 200 μL per well ± anti-CD3 (clone 145–2C11) and anti-CD28 (clone 37.51), 0.5 μg/mL each. At the end of incubation, cells were collected by centrifugation, and supernatant was used in experimental setups. The supernatant (10, 50 or 100 μL) was injected i.v. immediately before challenge with TNCB. Endogenous IL-10 and TGF-β were neutralized by i.p. injection of 100 μg anti-IL-10 and 50 μg anti-TGF-β (R&D Systems, Wiesbaden, Germany).

Cytokine levels were determined by sandwich ELISA using paired antibodies from the indicated companies: IL-10 (OptEIA Set Mouse IL-10, BD Biosciences) and TGF-β1 (TGFβ1 Emax®, Promega, Madison, WI).

Acknowledgements

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

This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 405, B16 and SFB 432, Z3) to A. H. Enk K. Mahnke and H. A. Lehr and the University of Mainz (MAIFOR) to A. H. Enk.

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

  • 6

    WILEY-VCH

  • 7

    WILEY-VCH

  • 1
    Enk, A. H. and Katz, S. I., Contact sensitivity as a model for T-cell activation in skin. J. Invest. Dermatol. 1995. 105: 80S–83S.
  • 2
    Bos, J. D. and Kapsenberg, M. L., The skin immune system: progress in cutaneous biology. Immunol. Today 1993. 14: 7578.
  • 3
    Cavani, A., Albanesi, C., Traidl, C., Sebastiani, S. and Girolomoni, G., Effector and regulatory T cells in allergic contact dermatitis. Trends Immunol. 2001. 22: 118120.
  • 4
    Okazaki, F., Kanzaki, H., Fujii, K., Arata, J., Akiba, H., Tsujii, K. and Iwatsuki, K., Initial recruitment of interferon-gamma-producing CD8+ effector cells, followed by infiltration of CD4+ cells in 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced murine contact hypersensitivity reactions. J. Dermatol. 2002. 29: 699708.
  • 5
    Bour, H., Peyron, E., Gaucherand, M., Garrigue, J. L., Desvignes, C., Kaiserlian, D., Revillard, J. P. and Nicolas, J. F., Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene. Eur. J. Immunol. 1995. 25: 30063010.
  • 6
    Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S., Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 1999. 162: 53175326.
  • 7
    Sakaguchi, S., Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 2004. 22: 531562.
  • 8
    Thornton, A. M. and Shevach, E. M., Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 2000. 164: 183190.
  • 9
    Green, E. A., Choi, Y., and Flavell, R. A., Pancreatic lymph node-derived CD4(+)CD25(+) Treg cells: highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity 2002. 16: 183191.
  • 10
    Laurie, K. L., Van Driel, I. R. and Gleeson, P. A., The role of CD4+CD25+ immunoregulatory T cells in the induction of autoimmune gastritis. Immunol. Cell Biol. 2002. 80: 567573.
  • 11
    Papiernik, M. and Banz, A., Natural regulatory CD4 T cells expressing CD25. Microbes Infect. 2001. 3: 937945.
  • 12
    Singh, B., Read, S., Asseman, C., Malmstrom, V., Mottet, C., Stephens, L. A., Stepankova, R. et al., Control of intestinal inflammation by regulatory T cells. Immunol. Rev. 2001. 182: 190200.
  • 13
    Dubois, B., Chapat, L., Goubier, A., Papiernik, M., Nicolas, J. F. and Kaiserlian, D., Innate CD4+CD25+ regulatory T cells are required for oral tolerance and inhibition of CD8+ T cells mediating skin inflammation. Blood 2003. 102: 32953301.
  • 14
    Dubois, B., Chapat, L., Goubier, A. and Kaiserlian, D., CD4+CD25+ T cells as key regulators of immune responses. Eur. J. Dermatol. 2003. 13: 111116.
  • 15
    Okazaki, F., Kanzaki, H., Fujii, K., Arata, J., Akiba, H., Tsujii, K. and Iwatsuki, K., Initial recruitment of interferon-gamma-producing CD8+ effector cells, followed by infiltration of CD4+ cells in 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced murine contact hypersensitivity reactions. J. Dermatol. 2002. 29: 699708.
  • 16
    Hwang, J. M., Yamanouchi, J., Santamaria, P. and Kubes, P., A critical temporal window for selectin-dependent CD4+ lymphocyte homing and initiation of late-phase inflammation in contact sensitivity. J. Exp. Med. 2004. 199: 12231234.
  • 17
    Groux, H., O'Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E. and Roncarolo, M. G., A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997. 389: 737742.
  • 18
    Mason, D. and Powrie, F., Control of immune pathology by regulatory T cells. Curr. Opin. Immunol. 1998. 10: 649655.
  • 19
    Liu, H., Hu, B., Xu, D. and Liew, F. Y., CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4. J. Immunol. 2003. 171: 50125017.
  • 20
    Schwarz, A., Maeda, A., Wild, M. K., Kernebeck, K., Gross, N., Aragane, Y., Beissert, S. et al., Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity. J. Immunol. 2004. 172: 10361043.
  • 21
    Maurer, M., Seidel-Guyenot, W., Metz, M., Knop, J. and Steinbrink, K., Critical role of IL-10 in the induction of low zone tolerance to contact allergens. J. Clin. Invest. 2003. 112: 432439.
  • 22
    Noble, K. E., Harkness, D. and Yong, K. L., Interleukin 10 regulates cellular responses in moncyte/endothelial cell co-cultures. Br. J. Haematol. 2000. 108: 497504.
  • 23
    Komura, K., Hasegawa, M., Hamaguchi, Y., Saito, E., Kaburagi, Y., Yanaba, K., Kawara, S. et al., Ultraviolet light exposure suppresses contact hypersensitivity by abrogating endothelial intercellular adhesion molecule-1 up-regulation at the elicitation site. J. Immunol. 2003. 171: 28552562.
  • 24
    Sasaki, M., Jordan, P., Houghton, J., Meng, X., Itoh, M., Joh, T. and Alexander, J. S., Transfection of IL-10 expression vectors into endothelial cultures attenuates alpha4beta7-dependent lymphocyte adhesion mediated by MAdCAM-1. BMC Gastroenterol. 2003. 3: 3.
  • 25
    Schwarz, A., Grabbe, S., Riemann, H., Aragane, Y., Simon, M., Manon, S., Andrade, S. et al., In vivo effects of interleukin-10 on contact hypersensitivity and delayed-type hypersensitivity reactions. J. Invest. Dermatol. 1994. 103: 211216.
  • 26
    Kondo, S., McKenzie, R. C. and Sauder, D. N., Interleukin-10 inhibits the elicitation phase of allergic contact hypersensitivity. J. Invest. Dermatol. 1994. 103: 811814.
  • 27
    Green, E. A., Gorelik, L., McGregor, C. M., Tran, E. H. and Flavell, R. A., CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells through TGF-β-TGF-β receptor interactions in type 1 diabetes. Proc. Natl. Acad. Sci. USA 2003. 100: 1087810883.
  • 28
    Lehr, H. A., Hübner, C., Nolte, D., Kohlschütter, A. and Messmer, K., Dietary fish oil blocks the microcirculatory manifestations of ischemia-reperfusion injury in striated muscle in hamsters. Proc. Natl. Acad. Sci. USA 1991. 88: 67266730.

Supporting Information

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

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