In this study, we have reported differences of peripheral blood DC subsets between sarcoidosis and atopic diseases using four-color flow cytometry, and have analyzed the adequacy of CD1a and CD141 as a marker for mDC1 and mDC2, respectively by the production of IL-12p40. Sarcoidosis patients showed decreased peripheral total and myeloid DC count with similar population of both CD1a+ mDC and CD141+mDC subsets compared to the control subjects. In contrast, patients with atopic diseases showed lower CD1a+mDC count and higher CD141+mDC count, which suggests the involvement of CD1a-mDCs and CD141+mDCs in Th2-polarity in atopic diseases.
In recent years, many studies have examined quantitative alternations of circulating blood DCs in patients with different pathological conditions, for example, Th1-mediated diseases such as viral infections and cancers, or Th2-mediated diseases such as bronchial asthma and atopic dermatitis
[18–20]. Most investigations have focused on the proportion of mDC and pDC subsets. After a report describing two further subsets in mDCs, mDC1s as a Th1-promoting mDC subtype and mDC2s as a Th2-promoting mDC subtype
, an increasing number of studies have been analyzing mDC1/mDC2 subsets using CD1c or CD1a, and CD141
[10, 11] has been reported. CD1a and CD1c are the subgroup of CD1, which is structurally and functionally similar to MHC class I and II molecules; however, CD1 presents lipids and glycolipids rather than polypeptides on DCs. CD1 has also evolved a unique path of intracellular trafficking, processing, and loading of lipid antigens. CD141, thrombomodulin, is a glycoprotein on the surface of endothelial cells, and activates protein C as a cofactor of thrombin in the anticoagulant pathway. The immunological function of CD141 on mDCs is unknown. Relatively few studies have analyzed human peripheral blood mDC1 and mDC2 subsets. According to previous studies
[10, 12] performed ex vivo, CD1a+mDCs or CD1c+mDCs are generally accepted as representing mDC1s and CD141+mDCs as representing mDC2s. Hata et al.
 succeeded in making mDC1s and mDC2s from freshly isolated circulating monocytes using either GM-CSF with IL-4 or IL-3 with IL-4, respectively. Furthermore, they confirmed the potential of CD1a as a marker for mDC1s through the differences in expression between mDC1 and mDC2 subsets. Based on these findings and results of previous reports
[10–12], we used CD1a and CD141 in this study as a marker of mDC1s and mDC2s, respectively.
Sarcoidosis is a multisystem disorder of unknown etiology characterized by non-caseating granulomas that are composed of epithelioid cells, fibroblasts, and several immune cells such as T cells and histiocytes/macrophages. Lungs, eyes and skin are the most affected organs. In sarcoidosis, lung T cells are shown to spontaneously release high levels of IL-2 and interferon γ
[22–24], and studies of T-cell clones from lung parenchyma
 and of bronchoalveolar lavage fluid (BALF)
[26–29] support the opinion of sarcoidosis as a Th1-mediated disease. Similarly, various clinical studies, genetic studies and animal models of allergic diseases support the notion that atopic diseases such as bronchial asthma, atopic dermatitis and allergic rhinitis are Th2-mediated disease.
In the present study, sarcoidosis patients showed decreased total DC and mDC counts compared to healthy controls. Previous studies have identified decreased numbers of total blood DCs and both subsets of CD11c+-myeloid DCs and CD11c--lymphoid DCs
. Another report showed that sarcoidosis patients tended to show decreased numbers of mDCs
. Our result was similar to these previous reports. In sarcoidosis, circulating blood DCs migrate into the affected tissues, contributing to the formation of sarcoid granulomas. This migration and accumulation into local inflammatory tissues may decrease the number of DCs circulating in the blood. Ota et al. showed the accumulation of DCs in the lymphocyte layer of sarcoid granulomas
We expected a predominance of CD1a+mDC count in peripheral blood in patients with sarcoidosis. Contrary to our expectation, no such differences in numbers of CD1a+mDCs and CD1a-mDCs were seen between sarcoidosis and controls. In sarcoidosis, the immunity of circulating blood does not always parallel that of locally affected organs, as seen from granuloma or BALF in lung tissue. For example, the CD4/CD8 ratio in BALF is usually high in sarcoidosis, but is not increased in peripheral blood. Given these findings, immunity of peripheral blood in patients with sarcoidosis is thought not to reflect the Th1/Th2 polarity. There are a few reports demonstrating the expression of CD1a on mDCs in the local inflammatory sites in sarcoidosis. An investigation in the BALF of inflammatory diseases showed an increase of CD1a-mDCs in sarcoidosis
. Another study of immunohistochemical investigation in muscular sarcoidosis demonstrated that CD1c+ mDCs scattered mainly in the lymphocyte layers of granulomas and the endomysium around the granulomas, while CD1c+mDCs expressed the mature DC marker CD83, but CD1a positive cells were not found by double immunostaining
. Since expression of surface markers on DCs is varied by the existing environment such as in organ tissue or in blood circulation, and also by the involved organs, precise evaluation is not easy. Further investigation is necessary to define the local immunity in sarcoidosis.
In the atopy group, numbers of total DC, mDC, pDC were all equivalent to controls. A previous study reported that total counts of circulating blood DCs were increased in patients with asthma
. Some reports have shown that allergen challenge causes a rapid decrease in the circulating mDC count
 and an accumulation of DCs in airway epithelium
[36–38] in patients with allergic asthma, while another report showed a trend toward to decreased number of circulating blood mDCs and a significant increase of pDCs in patients with atopic asthma
. The different disease states of the study participants may explain such contradictory results. Contrary to previous reports, which performed allergen challenge in patients with allergic asthma to invoke allergic inflammation, we examined stable atopic patients with no medication in order to exclude any influence of medications. This might have contributed to the unclear differentiation from controls. Actually, Upham et al. reported that decreases in circulating mDCs were most marked at 3 and 6 h post-allergen challenge, gradually returning to baseline levels
In the atopy group, CD1a+mDC count showed a significant decrease, while CD141+mDC count was significantly increased compared to controls. Yerkovich et al. reported that after allergen challenge the constitutive expression of CD141 on mDCs was increased in atopic individuals compared to non-atopic subjects
. The same group of investigators has also shown that CD141+mDCs are associated with Th2 polarizing response, whereas CD141-mDCs are associated with a mixed Th1/Th2 response. On the other hands, Jongbloed et al. reported that CD141+mDCs induce superior Th1 response compared to CD1c+DCs
, and the significance of CD141+mDCs is still in dispute. The results of the present study may support the theory that CD1+mDCs and CD141+mDCs represent mDC1s and mDC2s, respectively. The decreased mDC1s and increased mDC2s may reflect the Th2-skewed immunity in atopic disease.
When discussing the adequacy of CD1a and CD141 as a definite marker for mDC1s and mDC2s, respectively, the ability of sorted CD1a+mDCs and CD141+mDCs to induce Th1 and Th2, respectively, must be confirmed. However, few reports have examined this area, because these procedures need large amounts of peripheral blood, reducing clinical feasibility. In a previous report, monocyte-derived CD1a+mDCs produced high amounts of IL-12, a Th1-inducible cytokine, and induced Th1 when co-cultured with lymphocytes
. In our study, sorted CD1a+mDCs produced significantly higher levels of IL-12p40 compared to CD1a-mDCs. This result suggests that CD1a can offer a marker for human peripheral blood mDC1s. CD141+mDCs produced significantly less IL-12p40 compared to CD1a+mDCs but no differences in produced IL-12p40 levels were seen between CD141+mDCs and CD141-mDCs. CD141-mDC subset is probably composed of heterogeneous cell populations, and they are not always represent mDC2 subset. So, our results imply the possibility of CD141 as a marker for mDC2, however, further investigation is necessary to clarify the meaning of the expression of CD141 on mDCs as mDC2.
In the present study, we were unable to analyze CD1a/CD141 double positive or double negative mDCs due to technical limitations. There is almost no data about them in human peripheral blood, however, Bratke et al. showed that 45% of mDCs were CD1a positive and 78% of mDCs were CD141 positive in the analysis of BALF of never smokers
. It may be difficult to completely identify mDC1 and mDC2 only using CD1a and CD141. In future research, we feel it is necessary to find out the number and function of CD1a/CD141 double positive and double negative mDCs in human peripheral blood.