Potential role of blood dendritic cells in elicitation phase of contact hypersensitivity response – preliminary study

INTRODUCTION

Described in 1973, dendritic cells (DC) along with macrophages and B lymphocytes are regarded as one of the main groups of antigen-presenting cells (APC), and they represent less than 1% of peripheral blood mononuclear cells (PBMC) [1]. The distinction from similar cells, such as monocytes and macrophages, was based on their unique morphology [2, 3]. Dendritic cells’ population heterogeneity, in terms of cluster of differentiation markers (CD), function, and anatomic location, is derived from separate bone marrow (CD34+ stem cells) hematopoietic lineages [4]. It has been proven that DC are efficient stimulators of both T-cell proliferation in mixed leukocyte reactions and the antigen-specific T-cell response [5, 6]. There are three main subpopulations of dendritic cells in human blood – two myeloid dendritic cell subsets (mDC1 and mDC2) and one plasmacytoid subset (pDC) – but this division only includes their presumed origin [7]. All of these blood dendritic cell (BDC) subsets also vary in expression of Toll-like receptors, produced cytokines and response to pathogens (Table 1) [8]. Both subtypes of myeloid DC secrete IL-12; thus they are responsible for differentiation of Th1 cells from naïve T cells and production of interferon  (IFN-) and tumor necrosis factor  (TNF-) from natural killer cells (NK) and T cells [9]. This suggests that mDC may also recognize several bacterial components. On the other hand, pDC play a pivotal role in anti-viral defense, by producing interferons (IFN- and IFN-), although in chronic viral infections (HIV, HCV) their amount in the circulation decreases [10–13].
The role of dendritic cells in the contact hypersensitivity response seems to be crucial, yet poorly understood. Although some data clearly underline the key role of dendritic cells in the sensitization phase of CHS, there are only scarce data regarding their role in the elicitation (effector) phase. Bangert et al. [14] found not only CD1c+ dendritic cells (Langerhans cells) in inflammatory infiltrate, but also BDCA-2+, CD123+, CD45RA+, and CD62L+ plasmacytoid dendritic cells. Girard-Madoux et al. [15] revealed that deficiency in IL-10, which regulates maturation of DC and proinflammatory cytokine secretion, results in an excessive CHS response. The above facts suggest that BDC can be involved in contact dermatitis development and immune cutaneous surveillance.

OBJECTIVE

The aim of our study was to assess the role of human blood dendritic cells – plasmacytoid DC (BDCA-2, BDCA-4) and myeloid DC (BDCA1, BDCA3) – at the elicitation site of CHS.

MATERIAL AND METHODS

The study group consisted of 25 healthy volunteers with a mean age of 22.3 ±6.1 (12 females, 13 ma­les aged 18–36 years) with skin phototype II or III, as assessed by Fitzpatrick scoring system [16]. We selected these phototypes as they are found in the majority of the Central Europe population. They had no skin or other disease and were neither receiving nor taking any medication. Subjects exposed to the contact allergen diphenylcyclopropenone (DPCP) were excluded. Signed informed consent was taken from all participants before enrollment in the study. The study design was accepted by the local ethics committee of the Medical University of Lodz, no. RNN/48/2001/KE.
All the volunteers were sensitized with DPCP. Elicitation of CHS took place 3 weeks after exposure to DPCP. Responses were evaluated after 48 h by a subjective visual scoring system, and a 3 mm-punch skin biopsy was taken from the 3.2 mg DPCP elicitation site in each subject and was immunohistochemically stained with monoclonal mouse IgG1 antibodies directed against BDCA-1, BDCA-2, BDCA-3, and BDCA-4 (Miltenyi Biotec, Bergish Gladback, Germany) [17] and presence of plasmacytoid (pDC) and myeloid (mDC) blood dendritic cells was analyzed (Figures 1 and 2). In each specimen, the staining intensity of BDCA-1, BDCA-2, BDCA-3, and BDCA-4 was recorded by two independent observers in 6–8 adjacent high-power fields and graded as 0 (lack of cells in epidermis and/or dermis) or 1 (presence of any cells in epidermis and/or dermis). In each group the number of plasmacytoid DC (BDCA-2, BDCA-4) and myeloid DC (BDCA1, BDCA3) was counted, and then the percentage of BDC was evaluated in groups 1 and 2.

Statistical analysis

For statistical analysis the 2 test and Fisher’s exact test were applied. Values of p < 0.05 were considered statistically significant.

RESULTS

Based on the visual score of CHS the volunteers were divided into two groups: group 1 (0.00) where the CHS score was assessed as 0 (no reaction; n = 7) and group 2 (1.00) where the CHS score was assessed as 1 (any response noted; n = 18). The presence of pDC was observed in a significantly higher percentage of subjects from group 1 (60%) compared to group 2 (15%) (p = 0.043). mDC cells were present in a higher percentage in subjects from group 2 than those from group 1. However, the difference was not statistically significant (p > 0.05). The statistical analysis also revealed that presence of BDCA-1 does not depend on study group (p > 0.05), while presence of BDCA-4 does (p < 0.05). These results are shown in Figure 3.

DISCUSSION

Contact hypersensitivity is a T cell-mediated, delayed skin inflammatory process induced by skin exposure to low-weight haptens in sensitized individuals. For a long time it has been considered that antigen-specific CD4+ T cells are essential in development of CHS, although recent findings have shown that dendritic cells, both present in the skin (LC – interstitial cells) and migrating from the blood (pDC), orchestrate the immunological cutaneous response [18–20]. These potent leukocytes, normally absent in human skin, in response to various immunological stimuli, migrate into the epidermis and dermis, to regulate the response of T cells. Our previous study showed that UV radiation suppresses CHS and influences the Langerhans cell count [21]. Other factors affecting the DC count include microbial infection and stress [10]. Nevertheless, the exact role of pDC remains unclear. Plasmacytoid dendritic cells constitute a minor population of DC in the blood and can be found both in primary and secondary lymphoid organs. In normal conditions pDC can recognize pathogenic nucleic acids, but they are tolerant to self DNA/RNA released from the cells during apoptosis or necrosis. Breaching tolerance to self nucleic acids could lead to autoimmunity [22]. It includes forming self DNA complexes with anti-DNA antibodies like in systemic lupus erythematosus or aggregation of self DNA with the antimicrobial polypeptide LL37 described in psoriasis [23, 24].
In our study, we used immunohistochemical staining to determine inflammatory infiltrate composition in CHS. Similar studies on mDC and pDC balance have been conducted in atopic dermatitis (AD), lupus erythematosus and psoriasis. In AD there is an increase of pDC in peripheral blood, when in fact their amount in the epidermis is barely detectable. Furthermore, it could result in eczema herpeticum [25]. By contrast, in other chronic inflammatory skin disorders (psoriasis and lupus erythematosus) migration of pDC into damaged skin is significantly higher [26]. Moreover, abundant presence of pDC has been described in certain skin tumors such as basal cell carcinoma, melanoma and squamous cell carcinoma in situ [25]. In our previous study we found that BDC are present in normal skin (mDC in epidermis, pDC in dermis) [8]. In this study we observed that a positive CHS response is linked with a decrease of pDC and increasing number of mDC. It has been suggested that migration of pDC is mediated by a recently discovered adipokine – chemerin. Chemerin, first described in psoriatic skin lesions, seems to be an important chemoattractant which triggers an inflammatory response in damaged skin [27]. The lower migration rate of pDC in contact hypersensitivity is probably caused by insignificant or suppressed chemerin expression in the epidermis. Apart from chemerin, there are also other attractants, which could be responsible for engaging pDC, such as adenosine and anaphylatoxins C3a and C5a. However, their hypothetical role in the elicitation phase of CHS needs to be fully investigated [28, 29]. Based on our results, one may assume that the imbalance between pDC and mDC may be the key to understanding the effector phase of CHS and may clarify its accurate etiology.

ACKNOWLEDGMENTS

The study was funded by the National Center of Science grants no. NN401063236 and 2012/05/B/NZ5/01885 and the Medical University of Lodz, project no. 503/1-152-01/503-01.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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Submitted: 10 XII 2015
Accepted: 1 III 2016