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- Materials and methods
Background: Psoriasis vulgaris is a common chronic inflammatory dermatosis. Disorders in keratinocyte proliferation, differentiation, inflammation and immune dysregulation are the major factors implicated in the pathogenesis of psoriasis vulgaris.
Methods: The study was performed in skin specimens of 25 patients with psoriasis vulgaris and a control group of 10 individuals without a skin disease. Biopsy specimens from lesional and normal skin were analyzed by immunohistochemical method for expressions of Ki-67, Bcl-2, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NF-κB). In addition, densities of mast cell infiltration were also investigated.
Results: Ki-67 and TUNEL indexes and TNF-α and NF-κB expressions were significantly higher in psoriatic epidermis than in normal epidermis (p < 0.05). There was no significant difference at Bcl-2 reactivity between the normal and the psoriatic epidermis (p > 0.05); however, Bcl-2 staining intensity of lymphocytes was higher in psoriatic lesions than in normal dermis (p < 0.05). Additionally, the number of mast cells was significantly higher in psoriatic dermis than in normal skin (p < 0.05).
Conclusions: There were several complex factors involved in the pathogenesis of psoriasis. We conclude that cellular damage and apoptosis temporarily coincide with epidermal proliferation during the course of psoriatic hyperplasia.
Psoriasis vulgaris is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of the epidermis and also inflammatory infiltration in the epidermis and dermis. These processes are mainly driven by activated T cells or antigen-presenting cells, which release various chemokines and cytokines that signal the keratinocytes to hyperproliferate, leading to abnormal differentiation.1–7 Proliferation of keratinocytes is restricted by apoptotic cell death to maintain a constant thickness of epidermis.8 There are several pathways to induce apoptosis.
Bcl-2 and its homologue proteins modulating apoptotic process were shown in psoriasis.9 Apoptosis is also induced by various stimuli, including ultraviolet radiation, tumor necrosis factor alpha (TNF-α) and Fas ligand. TNF-α is a major mediator in pathogenesis of psoriasis and a multifunctional cytokine that mediates inflammation, immune response and apoptosis.10 It is a homodimer of 157-amino acid subunits produced primarily by activated macrophages but also by other cell types, including epidermal keratinocytes.11,12 TNF-α has been shown to induce cell death of tumors by apoptosis or necrosis, and it also potentially contributes to the accumulation of inflammatory cells seen in the epidermis and dermis.1,13,14 Much higher levels of TNF-α are found in psoriatic lesional skin than in normal skin.2,12,15–18
Nuclear factor kappa B (NF-κB) has been shown to have an inhibitory role for keratinocyte growth in several studies.19–21 TNF-α and other inflammatory cytokines all of which are NF-κB activators are abundantly expressed in psoriatic epidermis (PE) where the hyperplasia is one of the foremost characteristic. Thus, the role of NF-κB in epidermis is still not well understood.20
Mast cells are mononuclear, nonphagocytic, granular immune effector cells found in various organs including the gastrointestinal tract, lungs and skin. Dermal infiltrate of the psoriasis consist of lymphomononuclear cells and mast cells. Mast cells are capable of releasing a wide range of factors that regulate the inflammatory process in skin disorders including psoriasis.22–25
The purpose of this study is to elucidate the nature of cell kinetics in a PE on the basis of differences in reactivities of TNF-α, NF-κB, Ki-67 and Bcl-2 and in the number of mast cells between the normal epidermis (NE) and the PE dermis.
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- Materials and methods
There exist two major pathological steps in psoriasis: epidermal hyperproliferation with abnormal differentiation and inflammatory infiltration of epidermis and dermis.6,25 The growth pattern of psoriatic skin tissue differs from normal by the great decrease in transit time of cells from the basal cell layer to the uppermost of the squamous cell layer (approximately 7 days compared with 53 days).1 Ki-67 is a widely used marker to detect S, G2 and M phases of the cell cycle.6 Our results indicate that the staining intensity of Ki-67 was higher in the PE than in the NE (p < 0.01), which may be regarded as a major finding that points out epidermal hyperproliferation. The findings of this study are also in accordance with the observations that show Ki-67 staining as a notable indicator for PE.4,15,26,27
TUNEL method is reliably used to detect free 3′-OH DNA ends that occur during apoptosis.13 However, Kumar et al. and Bovenschen et al.15,16 examined PE using TUNEL method and hesitated about its reliability for PE, as TUNEL is based on in situ labeling of DNA breaks of 3′-OH ends that might occur not only during apoptosis but also in necrosis and mitosis. Although anti-apoptotic features of PE has been proven by a variety of studies, contradicting reports state an increase in positive cells with TUNEL method throughout all cell layers of PE.9,26–29 In our study, TUNEL apoptotic index was higher than NE compared with PE. However, classical apoptotic markers (i.e. condense chromatin, nucleus and nucleolar disintegration, extensive cytoplasmic vesiculation, apoptotic body and nuclear debris) were not remarkable. Furthermore, TUNEL-positive keratinocytes were observed in all layers of psoriatic dermis in this study. Thus, we speculate that necrosis, autophagic cells or actively dividing keratinocytes might be mistakenly labeled in PE and were not specific for apoptosis. Therefore, a marked discrepancy between cell death and proliferation in the PE should be considered.
Bcl-2 is a proto-oncogene localized in chromosome 18 and aims to protect permanent cells from apoptosis.28 Controversial labeling results with anti-Bcl-2 antibodies have been observed for patients with psoriasis vulgaris.9 Takahashi et al. and Hussein et al.3,9 concurrently reported a significant decrease in Bcl-2 in PE. In contrast, Wrone-Smith et al.30 reported that neither PE nor NE expresses Bcl-2. Although the difference was not statistically considerable, we found increased Bcl-2 expression in PE compared with NE. The staining intensity of Bcl-2 was extremely weak (about 5–10%) in both groups or even nonexistent in some of the samples
Our results suggest that excessive mitogenic stimuli may result psoriatic epidermal hyperplasia that promote an increase in the proliferative cell compartment rather than a loss of antiproliferative control. Bianchi et al.31 reported that Bcl-2 expression was lower in keratinocytes in lichen planus and psoriasis than in healthy controls and related this finding with increased keratinocyte apoptosis. They also stated that while neutrophils and macrophages do not express bcl-2, lymphocytes are capable of expressing Bcl-2.31
Our results suggest that increased lymphocyte survival because of prevention of apoptosis leads to chronic and relapsing character of psoriasis. However, this concept is not specific to psoriasis, and other chronic relapsing inflammatory dermatoses may also present with apoptotic block that results increased lymphocyte survival.5 It has been shown that TNF-α triggers interleukin (IL)-1, IL-6, IL-8 and NF-κB production.1,17,32 The interaction between cytokines and growth factors contributes to the hyperproliferation of keratinocytes, increased neovascularization and inflammation that is frequently observed in psoriasis vulgaris.17,20,32 The activation of NF-κB may play a key role in increased keratinocyte survival and epidermal hyperproliferation in psoriasis.32 In our study, TNF-α and NF-κB reactivities were significantly higher in PE. Increased TNF-α levels in PE pose as another key stimulus in the pathogenesis of disease. However, the effect of TNF-α appears to be multifaceted, as the role of NF-κB.21,32 We believe that in the setting of a chronic inflammatory state such as psoriasis, there is an imbalance between the anti-apoptotic role and the cell cycle inhibitory role of NF-κB whereby the scale is tipped toward protection against cell death in the context of a constitutive cytokine-rich inflammatory milieu. In cells, where NF-κB is induced by TNF-α, apoptosis may not occur. This allows for the increased epidermal thickness and hyperproliferation seen in PE. Further researches will be needed to expand these ideas and to confirm the potential benefit of NF-κB-targeted therapies in the treatment of psoriasis and other inflammatory and autoimmune disorders.19,21,32
Mast cells are important not only in allergic and late-phase reactions but also in inflammation and T-cell-mediated immunity. These cells are depicted as having the key role in pathogenesis of various inflammatory diseases by regulating the permeability of local vessels in response to a variety of endogenous and exogenous triggers.33 Thus, pro-inflammatory substances such as histamine, heparin and TNF-α released from mast cells may contribute significantly to the pathogenesis of psoriasis. In psoriasis, mast cells are most numerous just beneath the epidermis, enabling these cells to communicate with keratinocyte.22,23 We noted that number of mast cells was significantly higher in psoriatic dermis than in normal dermis. This result is consistent with the previous studies.22–24 These results indicate a potential initiator role of mast cells in the pathogenesis of psoriasis.
Psoriasis is a chronic inflammatory skin disorder that is currently not curable. Many modes of therapy exist, but the disease is often resistant to treatment. The relapses are frequent on cessation of medication. Although the cause of psoriasis is still unknown, complex interactions between altered keratinocyte proliferation and immune dysregulation are accused.4
Therefore, we conclude that psoriatic proliferation is activated by various stimuli that cause cellular damage coupled with apoptosis and, at the same time, initiation of an epidermal proliferation process. However, regulatory mechanism of psoriasis should be further investigated and yet to be defined.