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Immunophenotyping of dendritic cells in lesional, perilesional and distant skin of chronic plaque psoriasis
Cellular Immunology 269 (2011) 115–119
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Immunophenotyping of dendritic cells in lesional, perilesional and distant skin of chronic plaque psoriasis Hanan AlSaeid Alshenawy a,b,⇑, Eiman Adel Hasby a a b
Lecturer of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
a r t i c l e
i n f o
Article history: Received 20 November 2010 Accepted 15 March 2011 Available online 17 March 2011 Keywords: Dendritic cells Psoriasis Langerhan‘s cells Plasmacytoid dendritic cells Immunotherapy
a b s t r a c t Psoriasis affects 2.7% of the world’s population. The sequence of these events remains controversial. Because antigen presenting is necessary for T-cell activation, dendritic cells may be involved in the pathogenesis of psoriasis. To investigate their role, we examined immunophenotyping of different dendritic cells and their distribution and numbers in psoriasis patients. Immunohistochemistry of CD1a, CD11c, CD86 and BDCA2 were performed using paraffin-embedded tissue obtained from a total of 45 patients with psoriasis. Samples were taken from the lesion, perilesional and distant skin and normal skin obtained from 10 control cases. There were marked increase in the number of positive CD1a, CD11c, CD86 and BDCA2 cells in perilesional and the psoriatic skin when compared to the distant skin and they were the least in the normal control skin. So different dendritic cells subsets have a very important role in psoriasis pathogenesis especially in initiation of the plaque in the perilesional skin. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Psoriasis is a common inflammatory skin disease that may present in several different forms, but the most common (>80% of cases) is chronic plaque psoriasis, consisting of red, heavily scaled, well-demarcated plaques on elbows, knees, scalp, and lower back. On routine histology, plaques of psoriasis show abnormal proliferation and differentiation of epidermal keratinocytes. Currently, there is no cure for psoriasis, and although rarely fatal, the disease causes considerable morbidity and impairment of quality of life [1]. However, in the past 20 years, the clearance of psoriasis using targeted immunotherapies demonstrates the significant role of dendritic cells (DCs) as an important element in the pathogenesis of psoriasis [2]. Central to this concepts are the upstream activation of DCs, DC-induced cytokine production, and antigen presentation to type 1 T-cells. Subsequently, stimulated T-cells release tumor necrosis factor-alpha (TNF-a), interferon-gamma (IFN-c), and other cytokines [3]. Collectively, TNF-a and IFN-c induce a wide variety of responses including: signal transducer and activator of transcription 1 (STAT1) stimulation followed by expression of downstream response genes; activation of nuclear factor kappa B (NF-jB) signaling pathways; leukocyte migration to inflammatory regions; and ⇑ Corresponding author at: Department of Pathology, Tanta University, 25 Hamdy Gado street, Tanta, Egypt. E-mail address: [email protected] (H.A. Alshenawy). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.03.015
vasodilation that results in the erythema that is characteristic of psoriatic lesions [1]. DCs are key players in the immune mechanisms surrounding psoriasis, as well as other disease processes. DCs circulate in the bloodstream in immature forms, and migrate into peripheral tissues. Immature DCs function in antigen detection, uptake and processing, whereas mature DCs primarily function in antigen presentation as well as cytokine production. DCs have been linked to diseases of chronic inflammation including psoriasis, Sjögren’s syndrome, rheumatoid arthritis, systemic lupus erythematosus (SLE), and multiple sclerosis [4]. DC populations have been historically classified either spatially (circulating blood DCs, draining lymph node DCs, epidermal DCs, and dermal DCs) and by their presumed origin (myeloid DCs, plasmacytoid DCs [pDCs]) [5]. There are three cutaneous DC populations in the steady-state including epidermal Langerhan‘s cells (Langerin+, CD1a+), resident dermal myeloid DCs (CD11c+, CD1c+), pDCs (BDCA-2+, CD123+) and a recently identified few dermal population of CD14+DCs [5]. Understanding these DCs may lead to new therapeutic targets for augmenting or suppressing inflammation during human disease. DCs undergo a wide variety of modifications in becoming mature: increased expression of costimulatory molecules (CD40, CD80, CD86) and adhesion molecules (ICAM-1); the expression of CD83, which is marker of DC maturation; upregulation of surface MHC molecules; decreased expression of pro-inflammatory chemokine receptors; and downregulation of phagocytic and endocytic receptors [6].
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Langerhans cells (LCs, contain characteristic cytoplasmic Birbeck granules and express a variety of cell surface molecules (e.g. Lag, Langerin, CD1a, CD205). They could be more mature and active in some diseases expressing costimulatory molecules and maturation molecules [5]. LCs reside in the epidermis, where they serve as sentinels of the immune system, their responsibilities being to deliver information (antigen) to T cells within skin-draining lymph nodes. The ability of LCs to migrate from epidermis to regional lymph nodes is thus of critical importance to the induction of cutaneous immune responses. The initiation and regulation of LC mobilization and migration are orchestrated by cutaneous cytokines and chemokines [7]. A proportion of resident steady-state dermal DCs (CD11c+) appear to be able to proliferate in situ to maintain the baseline population. The major resident population in normal dermis is identified phenotypically with CD11c. In steady-state, CD11c+DCs are relatively immature with modest T cell stimulatory ability, but their immunostimulatory capacity can be greatly increased with DC maturing stimuli [8]. A specialized type of CD11c+DC is significantly increased in the epidermis and dermis of some inflammatory lesions in comparison to normal human skin; these cells express DC costimulatory markers (e.g. CD86) as well as the enzyme inducible nitric oxide synthase (iNOS), maturation markers (CD83) and increased levels of TNF-a [1]. Plasmacytoid DCs (pDCs) represent an uncommon cell type that morphologically resembles plasma cells. During pro-inflammatory states, pDCs are capable of generating type 1 interferons and migrating to inflamed tissue sites; type 1 interferon family members collectively serve a wide variety of functions including antiviral actions, the prevention of T-cell death, memory T-cell expansion, stimulation of DC maturation, and activation of NK cells [9]. A common phenotypic definition of pDCs is HLA-DR+CD11c-CD123+BDCA2+ [5]. Psoriatic plaques usually start as small papules enlarging outward to form well-demarcated typical lesions. The peripheral lesion of psoriatic plaques is thought to be more active than the center of the plaque. We have investigated the clues to early events at the center of the plaque and in the normal-appearing skin surrounding the psoriatic plaques (perilesional skin) to seek the subclinical phenomena preceding the formation of the psoriatic skin lesion, regarding DCs infiltrating the skin and compare this with the distant uninvolved and normal control skin.
under local anesthesia (1% lidocaine). Biopsies of psoriasis skin were taken within a lesion, 1 cm from the edge of the plaque border. Biopsies of perilesional skin were taken 2 cm beyond the plaque border while biopsies from univeloved skin were taken far distant from any psoriatic plaque by 10 cm at least. Also two punch biopsies were taken from each volunteer as control. Biopsies were placed immediately in 4% buffered formal saline for routine processing to paraffin sections and staining with hematoxylin and eosin. 2.3. Immunohistochemical study of DCs Sections (5 lm) were fixed and subjected to antigen retrieval using microwave oven. The endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide, and the slides were then treated with 10% normal horse or goat serum for 30 min. Incubation with primary antibodies was performed at 4 °C overnight. The primary antibodies used were: CD1a (clone SK9, Becton Dickinson), CD11c (BD Biosciences, San Diego, CA), CD86 1:80 (BD Biosciences), BDCA2 PE (BD Biosciences). Immunostaining was accomplished using appropriate ABC Vectastain Kit (Vector Laboratories, Burlingame, CA) per manufacturer’s instructions. After incubation with primary antibodies overnight at 4 °C, slides were washed with phosphate-buffered saline, incubated with secondary antibodies for 2 h at room temperature, and stained with an avidin–biotin method using diaminobenzidine. They were counterstained with hematoxylin then mounted and covered with cover glass slides and observed under a light microscope. Positively stained cells were counted in 50 consecutive low power fields (40 magnification), and the mean values were calculated for each tissue sample. The mean number of cells collectively in all the cases was calculated and presented as mean cell number/ square millimeter. 2.4. Statistical analysis Data were summarized as mean ± SD. Comparisons between groups were performed using the two-sided Wilcoxon signed rank test for paired data. A significance level of p 6 0.05 was used for all comparisons as a significant value. 3. Results 3.1. Characterization of epidermal LCs
2. Materials and methods 2.1. Patient studies Forty-five patients with chronic plaque psoriasis (35 male and 10 female; age range, 18–58 years; mean age, 40.9 years) and ten healthy volunteers (5 female and 5 male; age range, 20–55 years; mean age, 38.1 years) took part in the study. Patients had a chronic plaque psoriasis of moderate severity and activity with the mean psoriasis area and severity index (PASI) of 24.56 ± 8.28 (M ± SD) and the average percentage of skin involvement of 28.44 ± 19.54%. The mean duration of psoriasis calculated in months was 92.10 ± 76.46. Systemic and topical treatment was stopped 4 weeks before biopsy was taken. An informed written consent was obtained after complete description of the study from each participant. 2.2. Tissue sampling Three punch biopsies (6 mm) each of psoriatic lesions and perilesional skin and normal uninvolved distant skin were taken
The density and the number of CD1a-positive cells are increased in the epidermis of perilesional skin and modestly increased in the plaque skin compared to distant skin and all were much more than the normal control skin (Fig. 1A–C). The mean densities of epidermal CD1a+LCs (M ± SD) was 45 ± 12/mm2 in psoriatic lesion, 55 ± 23 in perilesional skin, 20 ± 12 in distant skin and 10 ± 7 LC/mm2 in normal control volunteers. There is a difference between the four groups but it was not significant (p = 0.3). 3.2. Characterization of CD11c+DCs There were many CD11c+DCs distributed in both the dermis and epidermis of psoriatic lesions (230 ± 76 and 50 ± 12, respectively), in perilesional skin, they were (250 ± 88 and 65 ± 19, respectively), while in distant skin they were (120 ± 50 and 30 ± 10, respectively) and in normal control they were (50 ± 23 and 20 ± 5, respectively). The difference between the psoriatic, perilesional, normal patient skin and normal control differ significantly both in the dermis and epidermis (p = 0.002 and 0.04, respectively) (Fig. 2A–C).
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Fig. 1. Immunohistochemical staining of CD1+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
3.3. Characterization of costimulatory molecules stained by CD86 A resting phenotype was observed in DCs accumulating in the distant skin of psoriasis patients and normal control cases (no much increase in CD86+DCs). In contrast, DCs in psoriatic plaques and perilesional lesions showed enhanced levels of CD86. CD86+DCs in both epidermis and dermis of the psoriatic lesions were (32 ± 17 and 50 ± 12, respectively), while in perilesional skin it was (45 ± 34 and 65 ± 23, respectively). Distant skin of psoriatic patients showed small number of CD86+CDs, it was (11 ± 10 and 15 ± 13, respectively) while in normal control, it was (10 ± 9 and 15 ± 12, respectively). The difference between the psoriatic lesion and perilesional skin and both the normal and control was significantly different in both epidermis and dermis (p = 0.03 and 0.009, respectively), (Fig. 3A–C). 3.4. 4- Charecterization of BDCA2 DCs BDCA2+DCs was detected in the mid and papillary dermis of psoriatic patients in mild increased density and number (12 ± 6), in perilesional skin there were more increase (34 ± 12) while in distant skin, it was (7 ± 2) and their number was very poor in normal control skin (2 ± 1). The difference was significantly different between the studied cases (p = 0.012) (Fig. 4A–C).All these results are summarized in Table 1. 4. Discussion DCs are a unique population of leukocytes that regulate immune responses and have the ability to interact with and activate T cells. It has been proposed that the preferential association between DCs and epidermal T-cells may lead to the stimulation
and subsequent clonal expansion of epidermal T-cells which are key players in the production of pro-inflammatory cytokines that have been implicated in psoriasis [1]. In this study, CD1a+ epidermal LCs were increased in number in the psoriatic skin compared with perilesional skin, but both are lesser that of the uninvolved distant skin which was much lesser when compared with the normal control skin with significant difference. This was in agree with Komine et al. [10] who reported the same results, while Gilleaudeau et al. [11] showed that CD1a+LCs were increased in psoriatic lesions compared to healthy controls and uninvolved skin of psoriatic patients, but there was no statistical difference in numbers between the healthy control and distant skin of psoriatic patients. On the other hand, Deguchi et al. [12] showed that the number of CD1a+LCs in psoriatic lesions was not significantly different from that of normal controls, while Oxholm et al. [13] found that the number of CD1a+LCs in the distant skin of psoriatic patients was significantly decreased compared to that in healthy controls. The reason for these conflicting results may be due to the different CD1a+LC numbers in the different stages or sites of psoriatic plaques. Zaba et al. [5] have found that LCs were similar in lesional and non-lesional epidermis, with a redistribution to the upper layers of the thickened psoriatic epidermis. These observations are supported by studies showing that LC migration in psoriasis is impaired, leading to retention of LCs in psoriatic inflammation. The role of LCs in the pathogenesis of psoriasis is poorly understood. Normally, LCs reside in the epidermis, where they serve as sentinels of the immune system, their responsibilities being to deliver antigens to T cells within skin-draining lymph nodes. The initiation and regulation of LC migration are orchestrated by cutaneous cytokines and chemokines. Many observations Marie et al. [7] and Komine et al. [10] including the present study, have demonstrated that psoriasis is associated with a considerable
Fig. 2. Immunohistochemical staining of CD11+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
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Fig. 3. Immunohistochemical staining of CD86+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
Fig. 4. Immunohistochemical staining of BDCA2+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
Table 1 Characterization of different DCs by different immunohistochemical markers.
CD1a CD11c CD86 BDCA2
Epidermis Dermis Epidermis Dermis
Psoriatic lesion
Perilesional skin
Uninvolved skin
Normal control
p Value
45 ± 12 50 ± 12 230 ± 76 32 ± 17 50 ± 12 12 ± 6
55 ± 23 65 ± 19 250 ± 88 45 ± 34 65 ± 23 34 ± 12
20 ± 12 30 ± 10 120 ± 50 11 ± 10 15 ± 13 7±2
10 ± 7 20 ± 5 50 ± 23 10 ± 9 15 ± 12 2±1
0.3 0.04 0.002 0.03 0.009 0.012
impairment of epidermal LC mobilization. These data provide clear evidence that psoriasis is characterized by very substantial changes in LC biology. In this work, it was found that, in the perilesional and lesional skin of psoriatic plaques, large numbers of CD11c+DCs were found in the dermis and along the epidermal basement membrane especially at the perilesional skin and this was in accordance with Komine et al. [10] and Zaba et al. [14] who found a 30-fold increase in CD11c+DCs in the dermis. They suggested that they may be derived from circulating DC-precursors migrating into the skin due to inflammatory and chemotactic signals. These DCs are another possible participant in the early psoriatic change, which we revealed to have a prominent existence in the perilesional psoriatic epidermis. In the current study, BDCA2+DCs (pDCs) were detectable mainly in the lesional and perilesional skin of psoriatic patients, but they were detected in very few numbers in the distant uninvolved skin, and in the normal control skin. PDCs are reported by Nestle et al. [9] to play a critical part in initiating the development of psoriatic lesions. In their results, there were many pDCs in the lesional pap-
illary dermis of patients with psoriasis, while in Komine et al. [10] and Guttman-Yassky et al. [15] studies, there were no marked increase in the frequency of these cells in chronic large plaque psoriasis. Julie et al. [16] demonstrated increased numbers of pDC in psoriasis skin lesions but are also present in the healthy skin of these same patients. Within the lesions they are located in the dermis and have an activated phenotype. In parallel, the number of circulating PDC is reduced in these patients, which favors a cutaneous recruitment. In studies of normal skin by Zaba et al. [8], there are a small number of pDCs while Julie et al. [16] demonstrated that there were no pDCs in normal skin of the control cases. Banchereau et al. [17] and Leanne et al. [18] showed that pDCs accumulate in the skin of psoriasis patients and become activated to produce IFN-a early during disease development. Furthermore, pDCs drive the activation and expansion of autoimmune T cells in prepsoriatic skin, leading to the development of psoriasis. So, pDCs in prepsoriatic skin may determine the onset of local autoimmune inflammation leading to disease formation. To study the activity profile of the DCs studied in this work, CD86 as one of the costimulatory molecules was evaluated. CD86 was of particular relevance because a reagent that blocks engagement of CD28 with its ligands like CD86 has been observed to improve psoriasis plaques skin in a clinical trial [10]. In this study, large numbers of DCs in the lesional and larger number in perilesional epidermis were positive for CD86, suggesting that many of the DCs in the lesional and perilesional epidermis express the costimulatory molecules required for antigen presentation and this was in agreement with Komine et al. [10] who reported the same results.
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Collectively, DCs have been shown to secrete numerous cytokines that are implicated in psoriasis including TNF-a, IFN-a, IL-12, IL-23, and IL-15. IFN-a, which is produced by pDCs that are present within psoriasis plaques, plays a key role in the stimulation and proliferation of T-cells as well as the formation of psoriatic skin. IL-12 and IL-23 trigger T-helper cell activation [19]. As illustrated by Zheng et al. [20] and Kagami et al. [21], IL-23 activates T-helper cells that subsequently produce IL-17 and IL-22; this latter cytokine contributes to dermal inflammation and epidermal hyperplasia characteristic of psoriasis. Similarly, IL-15 is a pro-inflammatory cytokine that induces T-cell proliferation as well as skin hyperplasia. In light of the immense cytokine network described, there exist numerous possible targets for anti-psoriatic immunologic therapies. In conclusion, the present findings suggest that the identification of novel DC subsets will help achieve a greater understanding of psoriasis as the mechanisms behind psoriasis depend upon interactions between DCs and T-cells; in targeting DC–T-cell interactions and associated cytokine networks, immunologic therapies would generally been effective in the treatment of psoriasis.
References [1] S.P. Jariwala, The role of dendritic cells in the immunopathogenesis of psoriasis, Arch. Dermatol. Res. 299 (2007) 359–366. [2] M.A. Lowes, A.M. Bowcock, J.G. Krueger, Pathogenesis and therapy of psoriasis, Nature 445 (2007) 866–873. [3] O. Boyman et al., The pathogenic role of tissue-resident immune cells in psoriasis, Trends Immunol. 28 (2007) 51–57. [4] P.D. Cravens, P.E. Lipsky, Dendritic cells, chemokine receptors, and autoimmune inflammatory diseases, Immunol. Cell Biol. 80 (2002) 497–505. [5] L.C. Zaba, J.G. Krueger, M.A. Lowes, Resident and ‘‘inflammatory’’ dendritic cells in human skin, J. Invest. Dermatol. 129 (2009) 302–308. [6] F. Chamian, J.G. Krueger, Psoriasis vulgaris: an interplay of T-lymphocytes, dendritic cells, and inflammatory cytokines in pathogenesis, Curr. Opin. Rheumatol. 16 (2004) 331–337. [7] C. Marie, M. Singh, R.J. Dearman, H.S. Young, I. Kimber, C.E.M. Griffiths, Impaired Langerhans cell migration in psoriasis, J. Exp. Med. 203 (2006) 953–960.
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[8] L.C. Zaba, J. Fuentes-Duculan, R.M. Steinman, J.G. Krueger, M.A. Lowes, Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages, J. Clin. Invest. 117 (2007) 2517–2525. [9] F.O. Nestle, C. Conrad, A. Tun-Kyi, B. Homey, M. Gombert, O. Boyman, et al., Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production, J. Exp. Med. 202 (2005) 135–143. [10] M. Komine, M. Karakawa, T. Takekoshi, N. Sakurai, Y. Minatani, H. Mitsui, Y. Tada, H. Saeki, A. Asahina, K. Tamaki, Early inflammatory changes in the ‘‘perilesional skin’’ of psoriatic plaques: is there interaction between dendritic cells and keratinocytes?, J Invest. Dermatol. 127 (2007) 1915–1922. [11] P. Gilleaudeau, M. Sullivan-Whalen, K.M. Wittkowski, K. Papp, M. Garovoy, W. Dummer, et al., Increase in TNF-alpha and inducible nitric oxide synthaseexpressing dendritic cells in psoriasis and reduction with efalizumab (antiCD11a), Proc. Nat. Acad. Sci. USA 102 (2005) 19057–19062. [12] M. Deguchi, S. Aiba, H. Ohtani, H. Nagura, H. Tagami, Comparison of the distribution and numbers of antigen-presenting cells among T-lymphocytemediated dermatoses: CD1a+, factor XIIIa+ and CD68+ cells in eczematous dermatitis, psoriasis lichen planus and graft-versus-host disease, Arch. Dermatol. Res. 294 (2002) 297–302. [13] A. Oxholm, P. Oxholm, B. Staberg, Reduced density of T6-positive epidermal Langerhans’ cells in uninvolved skin of patients with psoriasis, Acta. Dermatol. Venereol. 67 (1987) 8–11. [14] L.C. Zaba, I. Cardinale, P. Gilleaudeau, M. Sullivan-Whalen, M. Suarez Farinas, J. Fuentes-Duculan, et al., Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses, J. Exp. Med. 204 (2007) 3183–3194. [15] E. Guttman-Yassky, M.A. Lowes, J. Fuentes-Duculan, J. Whynot, I. Novitskaya, I. Cardinale, et al., Major differences in inflammatory dendritic cells and their products distinguish atopic dermatitis from psoriasis, J. Allergy Clin. Immunol. 119 (2007) 1210–1217. [16] C. Julie, L. Chaperot, D. Salameire, J.D. Domizio, C. Aspord, R. Gressin, J. MarieChristine, R. Marie-Jeanne, B. Jean-Claude, J. Plumas, L. Marie-Thérèse, Plasmacytoid dendritic cells and dermatological disorders: focus on their role in autoimmunity and cancer, Eur. J. Dermatol. 20 (2010) 16–23. [17] J. Banchereau, V. Pascual, A.K. Palucka, Autoimmunity through cytokineinduced dendritic cell activation, Immunity 20 (2004) 539–550. [18] M.J. Leanne, N.S. McNutt, J.G. Krueger, M.A. Lowes, Cytokine-producing dendritic cells in the pathogenesis of inflammatory skin diseases, J. Clin. Immunol. 29 (2009) 247–256. [19] B.J. Nickoloff, F.O. Nestle, Recent insights into the immunopathogenesis of psoriasis provide new therapeutic opportunities, J. Clin. Invest. 113 (2004) 1664–1675. [20] Y. Zheng et al., Interleukin-22, a TH17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis, Nature 445 (2007) 648–651. [21] S. Kagami, H.L. Rizzo, J.J. Lee, Y. Koguchi, A. Blauvelt, Circulating Th17, Th22, and Th1 cells are increased in psoriasis, J. Invest. Dermatol. 130 (2010) 1373–1383.
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Immunophenotyping of dendritic cells in lesional, perilesional and distant skin of chronic plaque psoriasis Hanan AlSaeid Alshenawy a,b,⇑, Eiman Adel Hasby a a b
Lecturer of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
a r t i c l e
i n f o
Article history: Received 20 November 2010 Accepted 15 March 2011 Available online 17 March 2011 Keywords: Dendritic cells Psoriasis Langerhan‘s cells Plasmacytoid dendritic cells Immunotherapy
a b s t r a c t Psoriasis affects 2.7% of the world’s population. The sequence of these events remains controversial. Because antigen presenting is necessary for T-cell activation, dendritic cells may be involved in the pathogenesis of psoriasis. To investigate their role, we examined immunophenotyping of different dendritic cells and their distribution and numbers in psoriasis patients. Immunohistochemistry of CD1a, CD11c, CD86 and BDCA2 were performed using paraffin-embedded tissue obtained from a total of 45 patients with psoriasis. Samples were taken from the lesion, perilesional and distant skin and normal skin obtained from 10 control cases. There were marked increase in the number of positive CD1a, CD11c, CD86 and BDCA2 cells in perilesional and the psoriatic skin when compared to the distant skin and they were the least in the normal control skin. So different dendritic cells subsets have a very important role in psoriasis pathogenesis especially in initiation of the plaque in the perilesional skin. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Psoriasis is a common inflammatory skin disease that may present in several different forms, but the most common (>80% of cases) is chronic plaque psoriasis, consisting of red, heavily scaled, well-demarcated plaques on elbows, knees, scalp, and lower back. On routine histology, plaques of psoriasis show abnormal proliferation and differentiation of epidermal keratinocytes. Currently, there is no cure for psoriasis, and although rarely fatal, the disease causes considerable morbidity and impairment of quality of life [1]. However, in the past 20 years, the clearance of psoriasis using targeted immunotherapies demonstrates the significant role of dendritic cells (DCs) as an important element in the pathogenesis of psoriasis [2]. Central to this concepts are the upstream activation of DCs, DC-induced cytokine production, and antigen presentation to type 1 T-cells. Subsequently, stimulated T-cells release tumor necrosis factor-alpha (TNF-a), interferon-gamma (IFN-c), and other cytokines [3]. Collectively, TNF-a and IFN-c induce a wide variety of responses including: signal transducer and activator of transcription 1 (STAT1) stimulation followed by expression of downstream response genes; activation of nuclear factor kappa B (NF-jB) signaling pathways; leukocyte migration to inflammatory regions; and ⇑ Corresponding author at: Department of Pathology, Tanta University, 25 Hamdy Gado street, Tanta, Egypt. E-mail address: [email protected] (H.A. Alshenawy). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.03.015
vasodilation that results in the erythema that is characteristic of psoriatic lesions [1]. DCs are key players in the immune mechanisms surrounding psoriasis, as well as other disease processes. DCs circulate in the bloodstream in immature forms, and migrate into peripheral tissues. Immature DCs function in antigen detection, uptake and processing, whereas mature DCs primarily function in antigen presentation as well as cytokine production. DCs have been linked to diseases of chronic inflammation including psoriasis, Sjögren’s syndrome, rheumatoid arthritis, systemic lupus erythematosus (SLE), and multiple sclerosis [4]. DC populations have been historically classified either spatially (circulating blood DCs, draining lymph node DCs, epidermal DCs, and dermal DCs) and by their presumed origin (myeloid DCs, plasmacytoid DCs [pDCs]) [5]. There are three cutaneous DC populations in the steady-state including epidermal Langerhan‘s cells (Langerin+, CD1a+), resident dermal myeloid DCs (CD11c+, CD1c+), pDCs (BDCA-2+, CD123+) and a recently identified few dermal population of CD14+DCs [5]. Understanding these DCs may lead to new therapeutic targets for augmenting or suppressing inflammation during human disease. DCs undergo a wide variety of modifications in becoming mature: increased expression of costimulatory molecules (CD40, CD80, CD86) and adhesion molecules (ICAM-1); the expression of CD83, which is marker of DC maturation; upregulation of surface MHC molecules; decreased expression of pro-inflammatory chemokine receptors; and downregulation of phagocytic and endocytic receptors [6].
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Langerhans cells (LCs, contain characteristic cytoplasmic Birbeck granules and express a variety of cell surface molecules (e.g. Lag, Langerin, CD1a, CD205). They could be more mature and active in some diseases expressing costimulatory molecules and maturation molecules [5]. LCs reside in the epidermis, where they serve as sentinels of the immune system, their responsibilities being to deliver information (antigen) to T cells within skin-draining lymph nodes. The ability of LCs to migrate from epidermis to regional lymph nodes is thus of critical importance to the induction of cutaneous immune responses. The initiation and regulation of LC mobilization and migration are orchestrated by cutaneous cytokines and chemokines [7]. A proportion of resident steady-state dermal DCs (CD11c+) appear to be able to proliferate in situ to maintain the baseline population. The major resident population in normal dermis is identified phenotypically with CD11c. In steady-state, CD11c+DCs are relatively immature with modest T cell stimulatory ability, but their immunostimulatory capacity can be greatly increased with DC maturing stimuli [8]. A specialized type of CD11c+DC is significantly increased in the epidermis and dermis of some inflammatory lesions in comparison to normal human skin; these cells express DC costimulatory markers (e.g. CD86) as well as the enzyme inducible nitric oxide synthase (iNOS), maturation markers (CD83) and increased levels of TNF-a [1]. Plasmacytoid DCs (pDCs) represent an uncommon cell type that morphologically resembles plasma cells. During pro-inflammatory states, pDCs are capable of generating type 1 interferons and migrating to inflamed tissue sites; type 1 interferon family members collectively serve a wide variety of functions including antiviral actions, the prevention of T-cell death, memory T-cell expansion, stimulation of DC maturation, and activation of NK cells [9]. A common phenotypic definition of pDCs is HLA-DR+CD11c-CD123+BDCA2+ [5]. Psoriatic plaques usually start as small papules enlarging outward to form well-demarcated typical lesions. The peripheral lesion of psoriatic plaques is thought to be more active than the center of the plaque. We have investigated the clues to early events at the center of the plaque and in the normal-appearing skin surrounding the psoriatic plaques (perilesional skin) to seek the subclinical phenomena preceding the formation of the psoriatic skin lesion, regarding DCs infiltrating the skin and compare this with the distant uninvolved and normal control skin.
under local anesthesia (1% lidocaine). Biopsies of psoriasis skin were taken within a lesion, 1 cm from the edge of the plaque border. Biopsies of perilesional skin were taken 2 cm beyond the plaque border while biopsies from univeloved skin were taken far distant from any psoriatic plaque by 10 cm at least. Also two punch biopsies were taken from each volunteer as control. Biopsies were placed immediately in 4% buffered formal saline for routine processing to paraffin sections and staining with hematoxylin and eosin. 2.3. Immunohistochemical study of DCs Sections (5 lm) were fixed and subjected to antigen retrieval using microwave oven. The endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide, and the slides were then treated with 10% normal horse or goat serum for 30 min. Incubation with primary antibodies was performed at 4 °C overnight. The primary antibodies used were: CD1a (clone SK9, Becton Dickinson), CD11c (BD Biosciences, San Diego, CA), CD86 1:80 (BD Biosciences), BDCA2 PE (BD Biosciences). Immunostaining was accomplished using appropriate ABC Vectastain Kit (Vector Laboratories, Burlingame, CA) per manufacturer’s instructions. After incubation with primary antibodies overnight at 4 °C, slides were washed with phosphate-buffered saline, incubated with secondary antibodies for 2 h at room temperature, and stained with an avidin–biotin method using diaminobenzidine. They were counterstained with hematoxylin then mounted and covered with cover glass slides and observed under a light microscope. Positively stained cells were counted in 50 consecutive low power fields (40 magnification), and the mean values were calculated for each tissue sample. The mean number of cells collectively in all the cases was calculated and presented as mean cell number/ square millimeter. 2.4. Statistical analysis Data were summarized as mean ± SD. Comparisons between groups were performed using the two-sided Wilcoxon signed rank test for paired data. A significance level of p 6 0.05 was used for all comparisons as a significant value. 3. Results 3.1. Characterization of epidermal LCs
2. Materials and methods 2.1. Patient studies Forty-five patients with chronic plaque psoriasis (35 male and 10 female; age range, 18–58 years; mean age, 40.9 years) and ten healthy volunteers (5 female and 5 male; age range, 20–55 years; mean age, 38.1 years) took part in the study. Patients had a chronic plaque psoriasis of moderate severity and activity with the mean psoriasis area and severity index (PASI) of 24.56 ± 8.28 (M ± SD) and the average percentage of skin involvement of 28.44 ± 19.54%. The mean duration of psoriasis calculated in months was 92.10 ± 76.46. Systemic and topical treatment was stopped 4 weeks before biopsy was taken. An informed written consent was obtained after complete description of the study from each participant. 2.2. Tissue sampling Three punch biopsies (6 mm) each of psoriatic lesions and perilesional skin and normal uninvolved distant skin were taken
The density and the number of CD1a-positive cells are increased in the epidermis of perilesional skin and modestly increased in the plaque skin compared to distant skin and all were much more than the normal control skin (Fig. 1A–C). The mean densities of epidermal CD1a+LCs (M ± SD) was 45 ± 12/mm2 in psoriatic lesion, 55 ± 23 in perilesional skin, 20 ± 12 in distant skin and 10 ± 7 LC/mm2 in normal control volunteers. There is a difference between the four groups but it was not significant (p = 0.3). 3.2. Characterization of CD11c+DCs There were many CD11c+DCs distributed in both the dermis and epidermis of psoriatic lesions (230 ± 76 and 50 ± 12, respectively), in perilesional skin, they were (250 ± 88 and 65 ± 19, respectively), while in distant skin they were (120 ± 50 and 30 ± 10, respectively) and in normal control they were (50 ± 23 and 20 ± 5, respectively). The difference between the psoriatic, perilesional, normal patient skin and normal control differ significantly both in the dermis and epidermis (p = 0.002 and 0.04, respectively) (Fig. 2A–C).
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Fig. 1. Immunohistochemical staining of CD1+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
3.3. Characterization of costimulatory molecules stained by CD86 A resting phenotype was observed in DCs accumulating in the distant skin of psoriasis patients and normal control cases (no much increase in CD86+DCs). In contrast, DCs in psoriatic plaques and perilesional lesions showed enhanced levels of CD86. CD86+DCs in both epidermis and dermis of the psoriatic lesions were (32 ± 17 and 50 ± 12, respectively), while in perilesional skin it was (45 ± 34 and 65 ± 23, respectively). Distant skin of psoriatic patients showed small number of CD86+CDs, it was (11 ± 10 and 15 ± 13, respectively) while in normal control, it was (10 ± 9 and 15 ± 12, respectively). The difference between the psoriatic lesion and perilesional skin and both the normal and control was significantly different in both epidermis and dermis (p = 0.03 and 0.009, respectively), (Fig. 3A–C). 3.4. 4- Charecterization of BDCA2 DCs BDCA2+DCs was detected in the mid and papillary dermis of psoriatic patients in mild increased density and number (12 ± 6), in perilesional skin there were more increase (34 ± 12) while in distant skin, it was (7 ± 2) and their number was very poor in normal control skin (2 ± 1). The difference was significantly different between the studied cases (p = 0.012) (Fig. 4A–C).All these results are summarized in Table 1. 4. Discussion DCs are a unique population of leukocytes that regulate immune responses and have the ability to interact with and activate T cells. It has been proposed that the preferential association between DCs and epidermal T-cells may lead to the stimulation
and subsequent clonal expansion of epidermal T-cells which are key players in the production of pro-inflammatory cytokines that have been implicated in psoriasis [1]. In this study, CD1a+ epidermal LCs were increased in number in the psoriatic skin compared with perilesional skin, but both are lesser that of the uninvolved distant skin which was much lesser when compared with the normal control skin with significant difference. This was in agree with Komine et al. [10] who reported the same results, while Gilleaudeau et al. [11] showed that CD1a+LCs were increased in psoriatic lesions compared to healthy controls and uninvolved skin of psoriatic patients, but there was no statistical difference in numbers between the healthy control and distant skin of psoriatic patients. On the other hand, Deguchi et al. [12] showed that the number of CD1a+LCs in psoriatic lesions was not significantly different from that of normal controls, while Oxholm et al. [13] found that the number of CD1a+LCs in the distant skin of psoriatic patients was significantly decreased compared to that in healthy controls. The reason for these conflicting results may be due to the different CD1a+LC numbers in the different stages or sites of psoriatic plaques. Zaba et al. [5] have found that LCs were similar in lesional and non-lesional epidermis, with a redistribution to the upper layers of the thickened psoriatic epidermis. These observations are supported by studies showing that LC migration in psoriasis is impaired, leading to retention of LCs in psoriatic inflammation. The role of LCs in the pathogenesis of psoriasis is poorly understood. Normally, LCs reside in the epidermis, where they serve as sentinels of the immune system, their responsibilities being to deliver antigens to T cells within skin-draining lymph nodes. The initiation and regulation of LC migration are orchestrated by cutaneous cytokines and chemokines. Many observations Marie et al. [7] and Komine et al. [10] including the present study, have demonstrated that psoriasis is associated with a considerable
Fig. 2. Immunohistochemical staining of CD11+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
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Fig. 3. Immunohistochemical staining of CD86+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
Fig. 4. Immunohistochemical staining of BDCA2+DCs in psoriasis skin (A), perilesional skin (B) and distant uninvolved skin (C) [strept avidin–biotin-DAB].
Table 1 Characterization of different DCs by different immunohistochemical markers.
CD1a CD11c CD86 BDCA2
Epidermis Dermis Epidermis Dermis
Psoriatic lesion
Perilesional skin
Uninvolved skin
Normal control
p Value
45 ± 12 50 ± 12 230 ± 76 32 ± 17 50 ± 12 12 ± 6
55 ± 23 65 ± 19 250 ± 88 45 ± 34 65 ± 23 34 ± 12
20 ± 12 30 ± 10 120 ± 50 11 ± 10 15 ± 13 7±2
10 ± 7 20 ± 5 50 ± 23 10 ± 9 15 ± 12 2±1
0.3 0.04 0.002 0.03 0.009 0.012
impairment of epidermal LC mobilization. These data provide clear evidence that psoriasis is characterized by very substantial changes in LC biology. In this work, it was found that, in the perilesional and lesional skin of psoriatic plaques, large numbers of CD11c+DCs were found in the dermis and along the epidermal basement membrane especially at the perilesional skin and this was in accordance with Komine et al. [10] and Zaba et al. [14] who found a 30-fold increase in CD11c+DCs in the dermis. They suggested that they may be derived from circulating DC-precursors migrating into the skin due to inflammatory and chemotactic signals. These DCs are another possible participant in the early psoriatic change, which we revealed to have a prominent existence in the perilesional psoriatic epidermis. In the current study, BDCA2+DCs (pDCs) were detectable mainly in the lesional and perilesional skin of psoriatic patients, but they were detected in very few numbers in the distant uninvolved skin, and in the normal control skin. PDCs are reported by Nestle et al. [9] to play a critical part in initiating the development of psoriatic lesions. In their results, there were many pDCs in the lesional pap-
illary dermis of patients with psoriasis, while in Komine et al. [10] and Guttman-Yassky et al. [15] studies, there were no marked increase in the frequency of these cells in chronic large plaque psoriasis. Julie et al. [16] demonstrated increased numbers of pDC in psoriasis skin lesions but are also present in the healthy skin of these same patients. Within the lesions they are located in the dermis and have an activated phenotype. In parallel, the number of circulating PDC is reduced in these patients, which favors a cutaneous recruitment. In studies of normal skin by Zaba et al. [8], there are a small number of pDCs while Julie et al. [16] demonstrated that there were no pDCs in normal skin of the control cases. Banchereau et al. [17] and Leanne et al. [18] showed that pDCs accumulate in the skin of psoriasis patients and become activated to produce IFN-a early during disease development. Furthermore, pDCs drive the activation and expansion of autoimmune T cells in prepsoriatic skin, leading to the development of psoriasis. So, pDCs in prepsoriatic skin may determine the onset of local autoimmune inflammation leading to disease formation. To study the activity profile of the DCs studied in this work, CD86 as one of the costimulatory molecules was evaluated. CD86 was of particular relevance because a reagent that blocks engagement of CD28 with its ligands like CD86 has been observed to improve psoriasis plaques skin in a clinical trial [10]. In this study, large numbers of DCs in the lesional and larger number in perilesional epidermis were positive for CD86, suggesting that many of the DCs in the lesional and perilesional epidermis express the costimulatory molecules required for antigen presentation and this was in agreement with Komine et al. [10] who reported the same results.
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Collectively, DCs have been shown to secrete numerous cytokines that are implicated in psoriasis including TNF-a, IFN-a, IL-12, IL-23, and IL-15. IFN-a, which is produced by pDCs that are present within psoriasis plaques, plays a key role in the stimulation and proliferation of T-cells as well as the formation of psoriatic skin. IL-12 and IL-23 trigger T-helper cell activation [19]. As illustrated by Zheng et al. [20] and Kagami et al. [21], IL-23 activates T-helper cells that subsequently produce IL-17 and IL-22; this latter cytokine contributes to dermal inflammation and epidermal hyperplasia characteristic of psoriasis. Similarly, IL-15 is a pro-inflammatory cytokine that induces T-cell proliferation as well as skin hyperplasia. In light of the immense cytokine network described, there exist numerous possible targets for anti-psoriatic immunologic therapies. In conclusion, the present findings suggest that the identification of novel DC subsets will help achieve a greater understanding of psoriasis as the mechanisms behind psoriasis depend upon interactions between DCs and T-cells; in targeting DC–T-cell interactions and associated cytokine networks, immunologic therapies would generally been effective in the treatment of psoriasis.
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