Coexistence of Langerhans cells activation and immune cells infiltration in progressive nonsegmental vitiligo
2013; Elsevier BV; Volume: 73; Issue: 1 Linguagem: Inglês
10.1016/j.jdermsci.2013.09.004
ISSN1873-569X
AutoresSaori Itoi, Atsushi Tanemura, Yorihisa Kotobuki, Mari Wataya‐Kaneda, Daisuke Tsuruta, Masamitsu Ishii, Ichiro Katayama,
Tópico(s)Cell Adhesion Molecules Research
ResumoVitiligo vulgaris (VV) is an acquired hypomelanotic disease characterized by loss of melanocyte that is involved in 0.5–2% of the world population. Since it is well known that VV is occasionally associated with autoimmune thyroid disease and systemic lupus erythematosus, the autoimmune theory is considered as the most suitable to explain its pathogenesis [[1]Cunliffe W.J. Hall R. Newell D.J. Stevenson C.J. Vitiligo, thyroid disease and autoimmunity.Br J Dermatol. 1968; 80: 135-139Crossref PubMed Scopus (224) Google Scholar]. Recently, interplay among immune factors has been reviewed in terms of vitiligo pathophysiology [[2]Passeron T. Ortonne J.P. Activation of the unfolded protein response in vitiligo: the missing link?.J Invest Dermatol. 2012; 132: 2502-2504Crossref PubMed Scopus (31) Google Scholar]. For better understanding of the cellular immunity to melanocyte, we consider that it is important to assess comprehensive infiltration pattern of cells in vitiligo skin. In this study, we carried out immunohistochemistry for immune-competent cells and performed a confocal microscopic analysis for Langerhans cells (LCs) on lesional skin (LS), leading edge (LE), and non-involved lesion (NL). Skin biopsy specimens were obtained from 6 nonsegmental vitiligo patients in progressive state and those from 1 psoriasis patient and 3 normal individuals.Langerhans cells seemed to be more expanded and moved to the upper layer in LS, suggesting cell activation. The cytotoxic immune-response mediated by antigens presentation to CD8+ T cells may also be more activated in LS (Fig. 1A ). While mean cells size of LC was not differed among 3 lesions, both of those dendrite length and number tend to be increased in order of LS, LE, and NL of 6 VV patients (Fig. 1B). Next, we assessed and compared several types of skin infiltrating immune-cells number. The summary is shown in Table 1. The number of CD8+ cells, CD4+IL17A+ cells, and LCs were significantly increased in LS compared to NL. Vitiligo skin with higher number of infiltrating cells showed the more evident morphological alteration in Langerhans cells, indicating the coexistence of LC activation and the more intense infiltration especially of CD8+ T cells, Th17 cells, and dermal dendritic cells. The number of CD1a+CD207− dermal dendritic cell was also significantly increased in LS compared to NL, indicating occurrence of melanocyte-related antigens trafficking in LS. Instead, CD11c+ myeloid dendritic cells were more abundant in LE compared to LS and NL. CD56+ natural killer cells in LS and LE were sparse but more than NL. Foxp3+ T cells were similarly infiltrated in all of fields inconsistent with another study which emphasized decrease in functional regulatory T cells in vitiligo [[3]Elela M.A. Hegazy R.A. Fawzy M.M. Rashed L.A. Rasheed H. Interleukin 17, Interleukin 22 and Foxp3 expression in tissue and serum of non-segmental vitiligo: a case-controlled study on eighty-four patients.Eur J Dermatol. 2013; ([Epub ahead of print])PubMed Google Scholar]. As for the other findings of LC activation in vitiligo lesion, ultrastructual analysis showed that there was the increased number of Birbeck's granules preferably localized nearby the nucleus of LCs localized at the basal layer (Fig. 1C, upper row). In addition, the indeterminate dendritic cells without premelanosomes and Birbeck's granules which also named as α-dendritic cells were noticeably detected instead of missing melanocytes correspondent with previous reports (Fig. 1C, lower row) [4Prignano F. Ricceri F. Bianchi B. Guasti D. Bonciolini V. Lotti T. et al.Dendritic cells: ultrastructural and immunophenotypical changes upon nb-UVB in vitiligo skin.Arch Dermatol Res. 2011; 303: 231-238Crossref PubMed Scopus (8) Google Scholar, 5Mishima Y. Kawasaki H. Pinkus H. Dendritic cell dynamics in progressive depigmentations. Distinctive cytokinetics of dendritic cells revealed by electron microscopy.Arch Dermatol Forsch. 1972; 243: 67-87Crossref PubMed Scopus (59) Google Scholar].Table 1Result of immunohistochemical analysis for immune competent cells infiltration. Data represented as mean number of cells and SD. Statistical analysis for comparison was performed by using unpaired t-test.MelanACD8Foxp3CD4CD4+ IL17A+Epi. CD1aCD11cAHRHLA-DRCD123Der. CD1a+ CD207−CD56LS (n = 6)1.7±3.5**p-Value<0.05.39.5±22.7*p-Value<0.01.10.2±3.416.5±3.815.6±6.3*p-Value<0.01.33.5±10.0*p-Value<0.01.3.7±3.215.8±11.531.1±21.111.6±5.514.4*p-Value<0.01.4.5LE (n = 6)8.5±3.6**p-Value<0.05.19.9±15.4*p-Value<0.01.9.16±3.414.5±4.210.3±2.822.6±7.1*p-Value<0.01.17.3±8.3*p-Value<0.01.12.0±9.226.5±15.113.2±4.010.24.5NL (n = 6)19.2±7.814.3±10.59.1±3.310.7±3.35.2±1.419.2±3.95.8±3.77.8±2.318.1±8.610.3±3.37.61.75Psoriasis (n = 1)41.337.7*p-Value<0.01.18.332.3*p-Value<0.01.28.3**p-Value<0.05.46.7**p-Value<0.05.24.7**p-Value<0.05.132811.3ND2Normal (n = 3)17.8±2.27.8±2.47.9±3.212.3±2.18.5±1.05.9±0.95.88±1.17.8±3.316.3±9.27.9±2.500ND represent not done.* p-Value < 0.01.** p-Value < 0.05. Open table in a new tab As immune-related pathogenesis for nonsegmental vitiligo, recent growing evidences showed not only cytotoxic T cells but also association with Th17 cells to melanocyte dysfunction and disappearance in cooperation with residual skin components [[6]Kotobuki Y. Tanemura A. Yang L. Itoi S. Wataya-Kaneda M. Murota H. et al.Dysregulation of melanocyte function by Th17-related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris.Pigment Cell Melanoma Res. 2012; 25: 219-230Crossref PubMed Scopus (112) Google Scholar]. Other publications reported participation in natural killer cells and regulatory T cells in vitiligo [3Elela M.A. Hegazy R.A. Fawzy M.M. Rashed L.A. Rasheed H. Interleukin 17, Interleukin 22 and Foxp3 expression in tissue and serum of non-segmental vitiligo: a case-controlled study on eighty-four patients.Eur J Dermatol. 2013; ([Epub ahead of print])PubMed Google Scholar, 7Zhou L. Li K. Shi Y.L. Hamzavi I. Gao T.W. Henderson M. et al.Systemic ayalyses of immunophenotypes of peripheral T cells in non-segmental vitiligo: implication of defective natural killer T cells.Pigment Cell Melanoma Res. 2012; 25: 602-611Crossref PubMed Scopus (44) Google Scholar]. In this study, we showed obvious alteration of cell infiltration pattern among observed lesions. Differed infiltration was in not only cytotoxic T cells considered as main effector of melanocyte disappearance but also other immune competent cells such as dermal dendritic cells and Th17 cells. CD11c+ myeloid dendritic cells served as clearance of melanocyte debris were significantly increased in number in LE. It is commonly acceptable that melanocyte destruction caused by perforin produced from CD8 positive cytotoxic T cell, Anti-Mc antibody, and oxidative stress in the pathogenesis for vitiligo. We found increased infiltration of not only cytotoxic T cells but also Th17 cells and dermal dendritic cells and activation of LC in progressive vitiligo skin. Based upon this result and previous reports regarding local immune-milieu in nonsegmental vitiligo, we suggest a comprehensive pathogenesis for autoimmune vitiligo. There are some evidences to support direct melanocyte destruction by CD8 positive cytotoxic T cell following antigen presentation, pathogenic anti-melanocyte antibody and oxidative stress as local distress. In this study, we found the noticeable infiltration of kinds of inflammatory cells other than CD4+ and CD8+ T cells in active state vitiligo skin. The change of cell morphology suggests higher susceptibility to extrinsic pathogens and/or intrinsic preparation for innate immune response involving in TNF-α and IL-1β. Indeed, Moretti et al. reported cytokine imbalance in the vitiligo epidermis with increased expression of TNF-α and IL-6 [[8]Moretti S. Fabbri P. Baroni G. Berti S. Bani D. Berti E. et al.Keratinocyte dysfunction in vitiligo epidermis: cytokine microenvironment and correlation to keratinocyte apoptosis.Histol Histopathol. 2009; 24: 849-857PubMed Google Scholar]. Since LCs are commonly activated under IL-1α and TNF-α condition in case of contact hypersensitivity [[9]Kaplan D.H. Jenison M.C. Saeland S. Shlomchik W.D. Shlomchik M.J. Epidermal Langerhans cell-deficient mice develop enhanced contact hypersensitivity.Immunity. 2005; 23: 611-620Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar], it is conceivable that activated LCs may be an important factor on the occurrence of vitiligo as the interface of melanocyte-specific adoptive immunity cooperating with cytotoxic T cells and may also induce innate immunity in participation with Th17 cells. Following increased infiltration of CD11c+ myeloid dendritic cells and dermal CD1a+ dendritic cells in vitiligo skin can act as antigen trafficking to draining lymph nodes and can produce proinflammatory cytokines such as IL-6 and TNF-α leading to determine helper T cells polarization [[10]Fransen J.H. van der Vlag J. Ruben J. Adema G.J. Berden J.H. Hilbrands L.B. The role of dendritic cells in the pathogenesis of systemic lupus erythematosus.Arthritis Res Ther. 2010; 12: 207Crossref PubMed Scopus (67) Google Scholar]. Taken together with the effect of Th17 cell-related cytokines on surrounding keratinocyte and fibroblast [[6]Kotobuki Y. Tanemura A. Yang L. Itoi S. Wataya-Kaneda M. Murota H. et al.Dysregulation of melanocyte function by Th17-related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris.Pigment Cell Melanoma Res. 2012; 25: 219-230Crossref PubMed Scopus (112) Google Scholar], this positive feedback linage of local cytokines is possibly important for transient appearance of indeterminate dendritic cells and subsequent mature melanocyte disappearance. Given this idea on the underlying immunogenic mechanism, early therapeutic intervention of molecular targeting biologics is considerable for the treatment with progressive nonsegmental vitiligo.Grant supportThis work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the Ministry of Health, Labor and Welfare . Vitiligo vulgaris (VV) is an acquired hypomelanotic disease characterized by loss of melanocyte that is involved in 0.5–2% of the world population. Since it is well known that VV is occasionally associated with autoimmune thyroid disease and systemic lupus erythematosus, the autoimmune theory is considered as the most suitable to explain its pathogenesis [[1]Cunliffe W.J. Hall R. Newell D.J. Stevenson C.J. Vitiligo, thyroid disease and autoimmunity.Br J Dermatol. 1968; 80: 135-139Crossref PubMed Scopus (224) Google Scholar]. Recently, interplay among immune factors has been reviewed in terms of vitiligo pathophysiology [[2]Passeron T. Ortonne J.P. Activation of the unfolded protein response in vitiligo: the missing link?.J Invest Dermatol. 2012; 132: 2502-2504Crossref PubMed Scopus (31) Google Scholar]. For better understanding of the cellular immunity to melanocyte, we consider that it is important to assess comprehensive infiltration pattern of cells in vitiligo skin. In this study, we carried out immunohistochemistry for immune-competent cells and performed a confocal microscopic analysis for Langerhans cells (LCs) on lesional skin (LS), leading edge (LE), and non-involved lesion (NL). Skin biopsy specimens were obtained from 6 nonsegmental vitiligo patients in progressive state and those from 1 psoriasis patient and 3 normal individuals. Langerhans cells seemed to be more expanded and moved to the upper layer in LS, suggesting cell activation. The cytotoxic immune-response mediated by antigens presentation to CD8+ T cells may also be more activated in LS (Fig. 1A ). While mean cells size of LC was not differed among 3 lesions, both of those dendrite length and number tend to be increased in order of LS, LE, and NL of 6 VV patients (Fig. 1B). Next, we assessed and compared several types of skin infiltrating immune-cells number. The summary is shown in Table 1. The number of CD8+ cells, CD4+IL17A+ cells, and LCs were significantly increased in LS compared to NL. Vitiligo skin with higher number of infiltrating cells showed the more evident morphological alteration in Langerhans cells, indicating the coexistence of LC activation and the more intense infiltration especially of CD8+ T cells, Th17 cells, and dermal dendritic cells. The number of CD1a+CD207− dermal dendritic cell was also significantly increased in LS compared to NL, indicating occurrence of melanocyte-related antigens trafficking in LS. Instead, CD11c+ myeloid dendritic cells were more abundant in LE compared to LS and NL. CD56+ natural killer cells in LS and LE were sparse but more than NL. Foxp3+ T cells were similarly infiltrated in all of fields inconsistent with another study which emphasized decrease in functional regulatory T cells in vitiligo [[3]Elela M.A. Hegazy R.A. Fawzy M.M. Rashed L.A. Rasheed H. Interleukin 17, Interleukin 22 and Foxp3 expression in tissue and serum of non-segmental vitiligo: a case-controlled study on eighty-four patients.Eur J Dermatol. 2013; ([Epub ahead of print])PubMed Google Scholar]. As for the other findings of LC activation in vitiligo lesion, ultrastructual analysis showed that there was the increased number of Birbeck's granules preferably localized nearby the nucleus of LCs localized at the basal layer (Fig. 1C, upper row). In addition, the indeterminate dendritic cells without premelanosomes and Birbeck's granules which also named as α-dendritic cells were noticeably detected instead of missing melanocytes correspondent with previous reports (Fig. 1C, lower row) [4Prignano F. Ricceri F. Bianchi B. Guasti D. Bonciolini V. Lotti T. et al.Dendritic cells: ultrastructural and immunophenotypical changes upon nb-UVB in vitiligo skin.Arch Dermatol Res. 2011; 303: 231-238Crossref PubMed Scopus (8) Google Scholar, 5Mishima Y. Kawasaki H. Pinkus H. Dendritic cell dynamics in progressive depigmentations. Distinctive cytokinetics of dendritic cells revealed by electron microscopy.Arch Dermatol Forsch. 1972; 243: 67-87Crossref PubMed Scopus (59) Google Scholar]. ND represent not done. As immune-related pathogenesis for nonsegmental vitiligo, recent growing evidences showed not only cytotoxic T cells but also association with Th17 cells to melanocyte dysfunction and disappearance in cooperation with residual skin components [[6]Kotobuki Y. Tanemura A. Yang L. Itoi S. Wataya-Kaneda M. Murota H. et al.Dysregulation of melanocyte function by Th17-related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris.Pigment Cell Melanoma Res. 2012; 25: 219-230Crossref PubMed Scopus (112) Google Scholar]. Other publications reported participation in natural killer cells and regulatory T cells in vitiligo [3Elela M.A. Hegazy R.A. Fawzy M.M. Rashed L.A. Rasheed H. Interleukin 17, Interleukin 22 and Foxp3 expression in tissue and serum of non-segmental vitiligo: a case-controlled study on eighty-four patients.Eur J Dermatol. 2013; ([Epub ahead of print])PubMed Google Scholar, 7Zhou L. Li K. Shi Y.L. Hamzavi I. Gao T.W. Henderson M. et al.Systemic ayalyses of immunophenotypes of peripheral T cells in non-segmental vitiligo: implication of defective natural killer T cells.Pigment Cell Melanoma Res. 2012; 25: 602-611Crossref PubMed Scopus (44) Google Scholar]. In this study, we showed obvious alteration of cell infiltration pattern among observed lesions. Differed infiltration was in not only cytotoxic T cells considered as main effector of melanocyte disappearance but also other immune competent cells such as dermal dendritic cells and Th17 cells. CD11c+ myeloid dendritic cells served as clearance of melanocyte debris were significantly increased in number in LE. It is commonly acceptable that melanocyte destruction caused by perforin produced from CD8 positive cytotoxic T cell, Anti-Mc antibody, and oxidative stress in the pathogenesis for vitiligo. We found increased infiltration of not only cytotoxic T cells but also Th17 cells and dermal dendritic cells and activation of LC in progressive vitiligo skin. Based upon this result and previous reports regarding local immune-milieu in nonsegmental vitiligo, we suggest a comprehensive pathogenesis for autoimmune vitiligo. There are some evidences to support direct melanocyte destruction by CD8 positive cytotoxic T cell following antigen presentation, pathogenic anti-melanocyte antibody and oxidative stress as local distress. In this study, we found the noticeable infiltration of kinds of inflammatory cells other than CD4+ and CD8+ T cells in active state vitiligo skin. The change of cell morphology suggests higher susceptibility to extrinsic pathogens and/or intrinsic preparation for innate immune response involving in TNF-α and IL-1β. Indeed, Moretti et al. reported cytokine imbalance in the vitiligo epidermis with increased expression of TNF-α and IL-6 [[8]Moretti S. Fabbri P. Baroni G. Berti S. Bani D. Berti E. et al.Keratinocyte dysfunction in vitiligo epidermis: cytokine microenvironment and correlation to keratinocyte apoptosis.Histol Histopathol. 2009; 24: 849-857PubMed Google Scholar]. Since LCs are commonly activated under IL-1α and TNF-α condition in case of contact hypersensitivity [[9]Kaplan D.H. Jenison M.C. Saeland S. Shlomchik W.D. Shlomchik M.J. Epidermal Langerhans cell-deficient mice develop enhanced contact hypersensitivity.Immunity. 2005; 23: 611-620Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar], it is conceivable that activated LCs may be an important factor on the occurrence of vitiligo as the interface of melanocyte-specific adoptive immunity cooperating with cytotoxic T cells and may also induce innate immunity in participation with Th17 cells. Following increased infiltration of CD11c+ myeloid dendritic cells and dermal CD1a+ dendritic cells in vitiligo skin can act as antigen trafficking to draining lymph nodes and can produce proinflammatory cytokines such as IL-6 and TNF-α leading to determine helper T cells polarization [[10]Fransen J.H. van der Vlag J. Ruben J. Adema G.J. Berden J.H. Hilbrands L.B. The role of dendritic cells in the pathogenesis of systemic lupus erythematosus.Arthritis Res Ther. 2010; 12: 207Crossref PubMed Scopus (67) Google Scholar]. Taken together with the effect of Th17 cell-related cytokines on surrounding keratinocyte and fibroblast [[6]Kotobuki Y. Tanemura A. Yang L. Itoi S. Wataya-Kaneda M. Murota H. et al.Dysregulation of melanocyte function by Th17-related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris.Pigment Cell Melanoma Res. 2012; 25: 219-230Crossref PubMed Scopus (112) Google Scholar], this positive feedback linage of local cytokines is possibly important for transient appearance of indeterminate dendritic cells and subsequent mature melanocyte disappearance. Given this idea on the underlying immunogenic mechanism, early therapeutic intervention of molecular targeting biologics is considerable for the treatment with progressive nonsegmental vitiligo. Grant supportThis work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the Ministry of Health, Labor and Welfare . This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the Ministry of Health, Labor and Welfare . We thank Kenju Nishida and Eriko Nobuyoshi for their expert technical assistance.
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