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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

Background

Regulatory T cells (Tregs), together with tolerogenic dendritic cells (tDCs) are involved in maintaining peripheral tolerance. A recent report suggested both Tregs and tDCs may be pathogenic in granulomatous skin disorders.

Aim

To examined the expression of CD39 on granuloma-composing cells and Foxp3-positive Tregs in the skin in two representative granulomatous diseases, sarcoidosis and granuloma annulare (GA).

Methods

We immunohistologically examined expression of CD39 on granuloma-composing cells and expression of Foxp3 on CD4+ or CD25+ cells in fixed sections of lesional skin from 16 patients with sarcoidosis and five patients with GA.

Results

The granuloma-composing cells expressed CD39 in both sarcoidosis and GA. Significant numbers of CD4+ Foxp3+ Tregs were present diffusely throughout the granulomatous tissues in sarcoidosis, whereas Tregs in GA existed only at the peripheral lesion of palisading granulomatous tissue.

Conclusions

There was infiltration of increased numbers of Foxp3+ Tregs around the CD39+ granuloma-composing cells in both GA and sarcoidosis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

Regulatory T cells (Tregs) and tolerogenic dendritic cells (tDCs) are currently of interest in the pathogenesis of some human diseases, particularly in granulomatous skin disorders.[1, 2] Previous reports have suggested that the characteristic systemic granuloma arises as a fallback reaction to inefficient cellular immune processing, most often caused by impairment of myeloid dendritic cell function due to unknown causes.[3] With reference to Tregs in granulomatous skin disorders, the presence of Foxp3-positive Tregs in peripheral sarcoidosis granulomas, bronchoalveolar lavage fluid, and peripheral blood of patients with sarcoidosis was reported previously.[4] Concerning tolerogenic myeloid cells, it was reported that the indoleamine 2,3-dioxygenase (IDO) expression in myeloid cells is part of an integrated response to granuloma formation, which contributes to the onset of tolerance in adaptive immunity in sarcoidosis.[5]

CD39 is an ectonucleotidase that hydrolyses adenosine diphosphate (ADP) and adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is converted to adenosine by CD73 in the mouse.[6] Expression of CD39 and CD73 on murine Tregs suggests that adenosine could serve as an immunomodulatory component of the Treg suppressive repertoire.[7] CD39+ Tregs abolish ATP-dependent effects such as cellular toxicity and the maturation of dendritic cells (DCs).[6] In humans, the CD39-expressing T-regulatory effector memory cells are capable of suppressing interleukin (IL)-17 production.[8] In this study, we examined the expression of CD39 on granuloma-composing cells and Foxp3+ Tregs in the skin in two representative granulomatous diseases, sarcoidosis and granuloma annulare (GA).

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

Ethics approval

The study was approved by the ethics committee of Tohoku University Graduate School of Medicine, Sendai, Japan. All patients gave informed consent.

Samples and immunohistochemistry for Foxp3 and CD4, CD25 or CD39

We collected archival formalin-fixed paraffin wax-embedded skin specimens from 16 patients with sarcoidosis, 5 patients with granuloma annulare (GA), 4 patients with necrobiosis lipoidica (NL) and 5 patients with foreign-body granuloma from the Department of Dermatology at Tohoku University Graduate School of Medicine.

The 16 sarcoidosis samples and 5 GA samples were processed for double-staining with mouse monoclonal antibodies for human CD4 (Nichirei Co., Tokyo, Japan), human CD25 (Vector Inc., Burlingame, CA, USA), human CD39 (Abcam, Cambridge, UK), rabbit antihuman Foxp3 and immunoglobulin from an unimmunized rabbit (BioLegend Inc., San Diego, CA, USA), as described previously.[9] Mouse immunoglobulin G1 (IgG1), IgG2b and IgG2a (R & D Systems, Minneapolis, MN, USA) were used as isotype controls.

Briefly, formalin-fixed paraffin wax-embedded tissue samples were sectioned at 4 mm and dewaxed. After sections were autoclaved for antigen-retrieval treatment, they were blocked with goat serum for 10 min and then exposed to the primary antibodies at 4 °C overnight. Antibody binding was revealed via alkaline phosphatase–conjugated antirabbit immunoglobulin (Histofine SAB-AP (R) Kit; Nichirei Co.) for the anti-Foxp3 antibody and the immunoglobulin from an unimmunized rabbit, and via peroxidase-conjugated antimouse immunoglobulin (Histofine SAB-PO(M) Kits; Nichirei Co.) for the anti-CD4, anti-CD25a and anti-CD39 antibodies and their isotype controls. The anti-Foxp3 antibody was developed with liquid permanent red (Dako A/S, Glostrop, Denmark), whereas the other mouse antibodies were visualized with 3,3-diaminobenzidine tetrahydrochloride (Wako Pure Chemical Industries, Osaka, Japan).

Assessment of immunohistochemical staining

Staining of infiltrated lymphocytes was examined in at least five random representative fields from each section. The number of immunoreactive cells was counted using an ocular grid of 1 cm2 at a magnification of × 400 by two dermatologists, who were blinded to the original histology and who reviewed all of the slides independently. Data are expressed as the mean ± SD for each skin disorder.

Statistical analysis

For a single comparison of two groups, the Student t-test was used, and = 0.05 was considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

Expression of CD39 on granulomas in sarcoidosis and granuloma annulare

In sarcoidosis, CD39-positive cells were seen throughout the granuloma tissue (Fig. 1a,b), whereas in GA, CD39-positive cells were palisaded by lymphocytes (Fig. 1c,d).

image

Figure 1. Tissue samples from (a,b) patients with sarcoidosis and (c,d) patients granuloma annulare were stained with a combination of anti-CD39 and anti-Foxp3 antibodies, and developed with liquid permanent red (Foxp3; red) and 3,3-diaminobenzidine tetrahydrochloride (CD39; brown). Original magnification (a,c): ×100; (b,d) ×400.

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Foxp3+T cells in sarcoidosis and granuloma annulare

We found few Foxp3+ cell in areas of normal skin, consistent with our previous report.[9] Immunohistochemical staining showed an infiltration of substantial numbers of Foxp3+ cells in the noncaseating naked granuloma of sarcoidosis (Fig. 2a). Granuloma-composing cells were weakly positive CD4 cells. In contrast to sarcoidosis, Foxp3+ cells in GA were present around the palisading granuloma but had not infiltrated the granuloma (Fig. 2b). In addition, substantial numbers of Foxp3+ CD39+ cells were detected (Fig. 1b,d). The numbers of Foxp3+ cells were significantly higher in sarcoidosis (< 0.05) (Fig. 2c). By contrast, Foxp3+ was almost absent in both NL and foreign-body granuloma (data not shown).

image

Figure 2. Tissue samples from (a,b) patients with sarcoidosis and (c,d) patients granuloma annulare) were stained with a combination of (a) anti-CD4 and anti-Foxp3 antibodies or (b) anti-CD4 and anti-Foxp3 antibodies, developed with liquid permanent red (Foxp3; red) and 3,3-diaminobenzidine tetrahydrochloride for (a) CD39 or (b); CD25 (brown). (c) Total numbers of Foxp3+ regulatory T-cell (Treg) fraction cells in each field. Data are expressed as the mean ± SD of the Treg fractions in each skin disorder. *< 0.05.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

In this study, we examined the expression of the immunosuppressive cell-surface marker, CD39 ectonucleotidase (Entpd1), on granuloma-composing cells and Foxp3+ Tregs in the skin of patients with representative granulomatous diseases, sarcoidosis and GA. We found infiltration of increased numbers of Foxp3+ Tregs around the CD39+ granuloma-composing cells in both granulomatous skin disorders. Granuloma-composing cells were weakly positive CD4 cells, which suggested that these cells were mainly myeloid dendritic cells (mDCs) and macrophages, as described previously.

Sarcoidosis is a granulomatous disease of unknown aetiology, which is characterized by the formation of noncaseating granulomas in a variety of organs. Munro et al.[10] previously used the Kveim test as a model of sarcoidosis. Injection of Kveim suspension typically elicits a granulomatous response in a person with sarcoidosis, which is compositionally identical to sarcoid granuloma. Moreover, these authors also reported that patients with Kveim-positive sarcoidosis showed decreased delayed hypersensitivity reaction.[10, 11] These findings strongly suggested that patients with sarcoidosis had impaired cell-mediated immunity.[3, 10, 11] Interestingly, previous reports also suggested that patients with sarcoidosis could develop both haematopoietic malignancy and solid tumours.[12] By contrast, GA is rarely associated with solid tumours.[13] Indeed, Shimizu et al. found only eight cases of GA associated with solid tumours in the English literatures.[13] Therefore, we hypothesized that there was a difference, from an immunological point of view, in the profiles of granuloma-composing cells between sarcoidosis and GA. In this study, we examined this using immunohistochemical staining, focusing especially on immunosuppressive cells, namely Tregs and tDCs.

The roles of Tregs are of interest with regard to the pathogenesis of a number of human diseases, including granulomatous disease and skin tumours.[4, 14, 15] Depletion of CD4+ CD25+ Tregs results in the development of autoimmune disease and enhanced immune responses against alloantigens and tumour antigens.[2, 16] Downregulation of Tregs causes rejection of transplanted tumours by the host immune response.[17] By contrast, the high frequency of Tregs in patients with carcinomas reportedly contributes to lymphocyte dysfunction, leading to the suppression of anti-tumour immune responses.[7] Tregs are thus inextricably connected with immune tolerance and suppressed recognition of tumour antigens in tumour progression and recurrence. Together with Tregs, tDCs and immunosuppressive macrophages, such as myeloid-derived suppressor cells, are reported to contribute to establishing the tumour microenvironment.[18, 19]

Concerning the contribution of imuunosuppressive cells to granulomatous disease, Miyara et al. reported the presence of Foxp3+ Tregs in peripheral sarcoidosis granulomas, in bronchoalveolar lavage samples, and in the peripheral blood of patients with sarcoidosis.[4] They concluded that sarcoidosis is associated with global amplification of a Treg subset, whose activity is insufficient to control local inflammation.[4] More recently, von Bubnoff et al. reported the contribution to sarcoidosis of immunosuppressive DCs and macrophages expressing IDO.[5] They concluded that the IDO expression in myeloid cells is part of an integrated response of granuloma formation that contributes to the onset of tolerance in adaptive immunity. Furthermore, it was reported that Tregs from sarcoidosis-affected lung were mainly activated natural Tregs, but exhibited reduced repressor capacities despite high levels of IL-10 and transforming growth factor-β.[14] By contrast, the repressive capacity of Tregs from the peripheral blood of patients with sarcoidosis was not impaired compared with that from age-matched healthy donors. That report concluded that the suppressive function of Tregs in granulation tissue is abrogated by other immunological factors. In the present study, we found infiltration of Tregs into the granulation tissue, which was mainly composed of CD39+ myeloid DCs and macrophages. These observations imply that not only Foxp3+ Tregs, but also CD39+ myeloid cells might contribute to the onset of tolerance in the adaptive immunity to sarcoidosis.

CD39, also known as ectonucleoside triphosphate diphosphohydrolase 1, is an ectonucleotidase expressed on vascular endothelial cells, Tregs and tDCs.[18, 19] CD39 hydrolyses ATP and ADP to AMP, which is in turn converted to adenosine by CD73 in the mouse system.[20] Indeed, Dwyer et al.[21] suggested that differential expression of CD25 and CD39 on circulating CD4+ T cells can be used to distinguish between Tregs and other pathogenic cellular populations that secrete proinflammatory cytokines such as interferon-γ and IL-17. In the peripheral blood of patients with renal allografts, these proinflammatory cell populations are increased, and there is a concomitant decrease in CD4+ CD25+ CD39+ Tregs. Moreover, Loza et al.[22] previously reported that patients with minimally active lupus show almost no CD39+ Tregs, and the absence of CD39 is associated with reduced adenosine-mediated suppression of mitogen-stimulated T cells. In previous work, we also detected CD39+ Foxp3+ Tregs in the lesional skin of patients with invasive extramammary Paget disease, and concluded that the increase in immunosuppressive cells, including these functional Tregs, could be a compensatory mechanism in the immune system.[23] In the present study, we found that CD39 was present at the periphery of GA lesions, and that these lesions extended in a centrifugal pattern. By contrast, in sarcoidosis lesions, both CD39+ cells and Tregs were densely aggregated throughout the granuloma.

These observations suggest that the immune suppressive process involving Tregs might contribute to the pathological process of these granulomatous diseases. Although quantification of the expression of CD39 on Foxp3 is difficult, the immunosuppressive process involving CD39+ Tregs might contribute to the pathological process in both types of granulomatous disease.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

This study sheds light on the expression of CD39 by granuloma-composing cells and Tregs in noncaseating naked granuloma and palisading granuloma. We did not assess the function of these infiltrating Tregs and myeloid cells directly in this study. Further analysis of the mechanism of this phenomenon would offer fundamental insight into the establishment of sarcoidosis and GA.

What's already known about this topic
  • There is evidence suggesting an association between sarcoidosis and systemic malignancies.
  • Tregs, together with immunosuppressive macrophages, are involved in maintaining the tumour microenvironment.
  • Recent reports have suggested a pathological contribution of Tregs and granuloma-forming, myeloid tDCs and macrophages to sarcoidosis.
What does this study add?
  • Granuloma-forming cells in sarcoidosis are mainly positive for CD39.
  • Together with CD39+ cells in granuloma, Tregs were found in both sarcoidosis and GA.
  • Increased numbers of Foxp3+ Tregs were found diffusely scattered throughout the CD39+ granulomatous tissues in sarcoidosis, which may be related to the carcinogenesis of sarcoidosis.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

CPD questions

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

Learning objective

To demonstrate up-to date knowledge about peripheral tolerance in granulomatous disorders.

Question 1

From an immunological point of view, which of the following granulomatous diseases might be connected with carcinogenesis?

  1. Granuloma annulare.
  2. Sarcoidosis.
  3. Necrobiosis lipoidica.
  4. Foreign body granuloma.
  5. Granulomatous pigmented purpuric dermatitis.

Question 2

Which of the following T-cell subsets is connected with peripheral tolerance?

  1. T helper 17 cells.
  2. T helper 1 cells.
  3. T helper 9 cells.
  4. T helper 2 cells.
  5. Regulatory T cells.

Question 3

Which of the following cell-surface molecule hydrolyses adenosine diphosphate (ADP) and adenosine triphosphate (ATP) to adenosine monophosphate (AMP) to induce peripheral tolerance?

  1. CD62L.
  2. CD69.
  3. CD39.
  4. CD44.
  5. CD73.

Question 4

Which of the following granulomatous diseases was found to be composed of CD39+ cells?

  1. Necrobiosis lipoidica.
  2. Sarcoidosis.
  3. Elastolytic giant cell granuloma.
  4. Foreign body granuloma.
  5. Granulomatous pigmented purpuric dermatitis.

Question 5

Which of the following markers is known to be both necessary and sufficient for the development and function of CD4+ CD25+ regulatory T cells?

  1. The transcription factor T-bet.
  2. GATA3.
  3. Foxp3.
  4. Retinoic acid receptor-related orphan receptor (ROR)-γ.
  5. Signal transducer and activator of Transcription (STAT)6.

Instructions for answering questions

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. CPD questions
  10. Instructions for answering questions

This learning activity is freely available online at http://www.wileyhealthlearning.com/ced.

Users are encouraged to

  • Read the article in print or online, paying particular attention to the learning points and any author conflict of interest disclosures
  • Reflect on the article
  • Register or login online at http://www.wileyhealthlearning.com/ced. com and answer the CPD questions
  • Complete the required evaluation component of the activity

Once the test is passed, you will receive a certificate and the learning activity can be added to your RCP CPD diary as a self-certified entry.