Expression of CD1a and CD86 on scleroderma Langerhans cells

ARTICLE

Auteur(s) : Yong Xie, Xiaoyong Zhang, Yuji Inoue, Shouji Wakasugi, Takamitsu Makino, Hironobu Ihn

Department of Dermatology & Plastic and Reconstructive Surgery, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan

accepté le 19 Septembre 2007

Scleroderma is a connective tissue disease of unknown etiology that affects the microvasculature and connective tissue. It is characterized by thickening and fibrosis of the skin. Two types of scleroderma exist: localized scleroderma and systemic sclerosis (SSc).

In localized scleroderma, the lesions usually are limited to the skin and to the subcutaneous tissue beneath the cutaneous lesions. In SSc, in addition to involvement of the skin and the subcutaneous tissue, visceral lesions are present, leading to death in some patients.

Scleroderma is generally considered to have an autoimmune background because it is accompanied by various immunological abnormalities, such as antinuclear antibodies (ANA), anti-single stranded DNA (anti-ssDNA) antibodies, rheumatoid factor (RF), and lupus erythematosus cell phenomenon. Major antigens recognized by the ANA of patients with scleroderma are nuclear histones [1-9].

In the early stages of localized scleroderma, a striking accumulation of inflammatory cells among collagen bundles of the lower two-thirds of the reticular dermis can be seen. In addition, inflammatory cells are frequently seen among the fat cells of subcutaneous tissue, including lymphocytes, histiocytes, plasma cells and mast cells. The histologic appearance of the skin lesions in SSc is similar to that of localized scleroderma. However, in early lesions of systemic scleroderma the inflammatory reaction is less pronounced than in localized scleroderma, so that only a mild inflammatory infiltrate is present around the dermal vessels. Mononuclear cell infiltrates are much less common in SSc than in localized scleroderma, and are often less prominent [10].

Langerhans cells play an important role in the skin’s immune system. Little is known, however, about the antigen-presenting capacity of Langerhans cells in the context of skin inflammation. The presence of clustered Langerhans cells within the dermis has already been demonstrated in diverse inflammatory skin diseases, including psoriasis [11, 12], atopic dermatitis [12], lichen planus [13], allergic contact dermatitis [14] and delayed-type hypersensitivity [15]. However, the significance of the dermal Langerhans cells in such inflammatory lesions is still obscure, and it has not been referred to in recent reviews concerning dendritic cells (DCs) [16, 17].

DCs are the most capable antigen-presenting cells (APCs) required to initiate the immune responses. Langerhans cells are paradigmatic DCs that are present in the skin. Precursor Langerhans cells migrate to the epidermis via the hematogenous route. After an antigen capture, Langerhans cells migrate to the regional lymph nodes and convert into mature DCs that are capable of priming naive T cells [17]. During the process of maturation, Langerhans cells show increased expression of MHC class II molecules and costimulatory molecules B761 (CD80) and B7-2 (CD86) [18, 19].

The purpose of our experiment was to investigate the phenotypical characteristics of epidermal and dermal Langerhans cells and their spatial relationship with infiltrating lymphocytes in systemic scleroderma (SSc) and localized scleroderma.

Materials and methods

Patients

Cutaneous samples

Immunohistochemistry

Serial 4 μm thick sections were mounted on silane slides (Dako) and submitted to fixation, deparaffinization in xylene and dehydration through graded alcohols. The antigen was retrieved in 0.01 M citrate buffer (pH6.0) using a microwave oven for 15 minutes, for each antigen recovery of the molecules. After cooling at room temperature for 50 min, the slides were treated with 0.3% hydrogen peroxide in methanol (Merck, Germany) for 20 min to block endogenous peroxidases. Nonspecific staining was blocked with 5% normal horse serum for 45 minutes. Subsequently the slides were incubated with the primary antibodies diluted in horse serum albumin at 4 °C overnight in a humidity chamber. The samples were treated with biotinylated secondary antibodies for 30 minutes. After washing with PBS, the slides were incubated with ABC reagent for 60 minutes at room temperature. And then the slides were stained with DAB solution for 2-4 minutes under a microscope at room temperature. Finally the reaction was terminated by washing in distilled water. The slides were washed with PBS between each reaction step. Samples in which the primary antibody was omitted were used as a negative control. Positive labeling was identified by a brown staining around the cell membrane or by a brown color of the cytoplasm, according to the marker employed.

All sections were examined in an Olympus BX50 light microscope (Japan) and results were expressed as the mean count of cells per 10 high-power fields (HPF, 400×). Ten non-contiguous and non-overlapping microscopic fields were randomly chosen for cell counting in the epidermis or dermis. We judged a cell with a nucleus and clear immunoreactivity to be a positive cell and the positive cell numbers were estimated as a proportion of the lesion area.

The percentage of the stained cells with the specific antibody compared to the total cellular infiltration was counted.

Statistical analysis

Results

Summary of the immunohistochemical analysis

Quantitative assessment of CD1a and CD86 positive cells

Compared with the normal counterparts, the number of CD1a⁺ cells in localized scleroderma (figure 2B) increased slightly (p > 0.05) in the epidermis (5.3 ± 1.9/HPF), whereas dermal CD1a⁺ cells increased markedly (6.5 ± 2.0/HPF, p < 0.05). Dermal CD1a⁺ cells were frequently located in the clusters of lymphocytes (figure 1B). CD86⁺ cells can be observed in the dermis (figure 1E), showing a typical dendritic shape, but they were quite scarce in the epidermis (figure 2E).

Fewer CD1a⁺ cells (2.9±1.1/HPF, figure 1C) were detected in the dermis of SSc. There was statistical significance (p < 0.05) compared with localized scleroderma. A similar result was obtained comparing the number of CD86⁺ cells in SSc (1.0 ± 0.4/HPF) and localized scleroderma (2.8 ± 1.6/HPF) in the dermis (table 2). Compared with normal skin, the number of CD1a⁺ cells in SSc (figure 2C) decreased in the epidermis (2.0±0.8/HPF), but there was no statistical significance. No CD86⁺ cell was observed in the epidermis of SSc (table 3 and figure 2F).
Table 2 Quantitative assessment of CD1a and CD86 positive cells in dermis per HPF(×400)

Semiquantitative assessment of CD1a and CD86 positive cells

The CD1a mAb stained all seven lesions of SSc with a mean percentage of 7.8 ± 2.3% in the dermal infiltrate (figure 1C). The percentage of CD1a⁺ cells (figure 1B) in localized scleroderma (12.8 ± 3.1%) is higher than that in SSc and had statistical significance. In almost all the SSc sections, the CD1a expression showed weaker intensity than in the localized scleroderma sections. Both diseases had a much higher percentage than that in normal skin (2.7 ± 1.2%, p < 0.05).

The percentage of CD86⁺ cells was much lower than that of CD1a in both diseases and there was no statistical significance between localized scleroderma and SSc. But both diseases had significant differences in percentage compared to normal skin.
Table 4 The percentage of CD1a and CD86 positive cells in the infiltrate of dermis

Immunohistochemical analysis of CD208 cells

Discussion

Langerhans cells were originally defined as epidermal dendritic-shaped cells of neural origin by Paul Langerhans in 1868 [20], and their functions as APCs were identified later [16, 17]. Dermal Langerhans cells expressed B7-2 abundantly in inflamed skin. This predominant expression of B7-2 over B7-1 has already been demonstrated on CD68⁺ cells in human intestines [21, 22]. In vitro study of Langerhans cells suggested a crucial role of B7-2 in the induction of T cell proliferation with little dependence on B7-1 [23]. B7-2, therefore, could be an important molecule for costimulation by Langerhans cells.

The number of CD1a⁺ Langerhans cells in localized scleroderma was significantly higher number than in SSc, both in the dermis and in the epidermis. It suggested that CD1a⁺ Langerhans cells may play a more important role in localized scleroderma.

The numbers of CD1a⁺ Langerhans cells in the dermis of both localized scleroderma and SSc were higher than in normal skin. But in the epidermis, the number of CD1a⁺ cells of SSc was lower than that in normal skin. This indicated that, at least in the epidermis, Langerhans cells of SSc did not have a crucial role in the immuno-reaction.

CD86 was reported [24] to be predominantly expressed on cultured human Langerhans cells, and may transduce a primordial co-stimulatory signal into T cells. In mice, a co-stimulatory signal via the CD86 molecule has been reported to promote the differentiation from Th0 into Th2 lymphocytes [25]. Therefore, we focused our interest on the potential role of co-stimulatory molecules in the immunopathogenesis of scleroderma.

In this study we examined the CD86 expression on epidermal and derma Langerhans cells in scleroderma, and in normal skin as a control. In epidermis, CD86 were not expressed in almost all the samples, suggesting that B7-2 was not a functional molecule in the epidermis. CD86 was expressed in the dermis of lesional skin in all cases of localized scleroderma, in six of seven in SSc and two of four in normal skin. The number of CD86⁺ cells in the dermis of localized scleroderma was higher than that in SSc and normal skin and there was statistical significance. However, there was no significant difference in the number of CD86⁺ cells between SSc and normal skin. This indicates that B7-2 may play an important role in the pathogenesis of localized scleroderma.

In all cases, the ratio of CD86-positive Langerhans cells was much higher in dermis than in the epidermis, and Simon et al. have also observed the same tendency on B7/BB1 [26]. This increased expression rate is considered to be reasonable because Langerhans cells are thought to migrate from epidermis into dermis after in vivo activation by antigens.

We observed that the proportion of CD1a-positive Langerhans cells was higher than that of CD86-positive Langerhans cells both in the epidermis and in the dermis in all samples. Although almost all the CD86-positive cells in the epidermis were CD1a-positive Langerhans cells, some of the CD86-positive cells in the dermis did not express CD1a [27]. They might be macrophages, activated T cells and B cells, as those cells are known to express CD86, but we did not identify them. Previous studies have shown that the number of epidermal CD1a-positive cells decreased in systemic sclerosis [28, 29].

Mitra et al. [30] have demonstrated that anti-CD86 mAb significantly inhibited either SEB- or alloantigen-driven T-cell reactions interacted with dendritic cells (DDC) from normal skin. The study of Ohki et al. [27] was consistent with their findings, and suggested that CD86 may transduce a primordial co-stimulatory signal from the professional APC in skin, such as Langerhans cells and DDC, into T cells.

This study revealed that Langerhans cells may play an important role in the pathogenesis of scleroderma, especially in localized scleroderma. CD86 is predominantly expressed on dermal Langerhans cells in the lesional skin of localized scleroderma. Therefore, it may play an important role in the pathogenesis of localized scleroderma.

Acknowledgements

References

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