SCCA2-transfected human keratinocytes show increased secretion of IL-1α and IL-6, but not of TNF-α

Abstract

Psoriasis is a common skin disease characterized by epidermal hyperproliferation, capillary dilatation, and the presence of acute and chronic inflammatory cells in both the dermis and epidermis [1, 2]. A unifying hypothesis that accounts for the wide range of clinical characteristics of psoriasis highlights the role of cytokines in the etio-pathophysiology of this disease [8]. Epidermal cells, particularly keratinocytes, may play a central role in transmitting pro-inflammatory signals by the secretion of cytokines and growth factors [7]. The production of these factors, in particular IL-1 is usually very low, but can be induced significantly by various injurious agents, such as endotoxin, virus particles, tumor promoter or UV light [6, 7]. Both IL-1a and c are expressed in human keratinocytes, although the major isotype secreted is IL-1a [6]. IL-1 and TNF-a in their turn act with an autocrine mechanism on keratinocytes to induce the release of a cascade of other cytokines with pro-inflammatory or chemotactic activity (IL-6, IL-8) [3]. All of these mentioned cytokines have been detected in psoriatic lesions. In addition to these observations, Takeda et al. [11] reported that SCCA2, the member of serine proteinase inhibitor family (serpin) is the major inducible SCCA in psoriatic skin. They found that SCCA2 was expressed in stratum corneum, subset of spinous layer cells and dermal infiltrated cells [11]. The level of SCCA2 is significantly increased in the sera of psoriatic patients, compared with non-psoriatic individuals [4], and probably be due, at least in part, to the production of cytokines in psoriatic tissues. Moreover, recent study suggested that the expression of SCCA in keretinocytes may be participated in the growth of keratinocytes and their differentiation [11]. Since keratinocytes can secrete TNF-a, IL-1a, and IL-6, and since these molecules have been detected in psoriatic skin, the goal of this study was to test whether the expression of SCCA2 in human keratinocytes plays a role in modulating expression of these cytokines. Commercially available human epidermal keratinocytes (Kurabo, Japan) were grown in supplemented low calcium medium and were sub-cultured at 60–70% confluence to avoid differentiation. A SCCA2 construct consisting of the SCCA2 coding region cloned in to the p3XFLAG-myc-CMV-26 vector (Sigma) has been described elsewhere [12]. The vector [p3XFLAG-mycCMV-26 (Sigma)] without SCCA2 was transfected into the keratinocytes as a control. The SCCA2 construct or the control vector was transfected into human keratinocytes using lipofectamine reagent (Sigma) according to the manufacturer’s instructions. TNF-a, IL-1a, and IL-6 concentrations in the culture media were measured by using ELISA kits (R&D Systems Inc., USA) according to the manufacturer’s instructions. SCCA2 expression in transfected cell was determined by Western blot analysis using anti-SCCA antibody (Sigma). The statistical analysis was performed using Student’s t test, and P<0.05 was considered to be significant. The results are shown as mean ± standard deviation (SD). To show SCCA2 expression in cells transfected with the SCCA2 construct, cell lysates were subjected to Western blot analysis. SCCA2 expression was detected in cells transfected with the SCCA2 construct (Fig. 1a, lane 1) but not in cells transfected with vector alone (Fig. 1a, lane 2). Figure 2b shows a control blot using non-immunized mouse IgG. There is no conflict of interest in this manuscript.