Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro

BACKGROUND: thymus and activation-regulated chemokine (TARC)/CCL17 is a CC chemokine that selectively attracts Th2-type lymphocytes. Immunohistochemical analyses have revealed that TARC is expressed in the epidermal keratinocytes of atopic dermatitis (AD), suggesting TARC involvement in the pathogenesis of the disease. However, keratinocyte TARC production has been described only in the transformed keratinocyte cell line HaCaT. OBJECTIVE: to examine TARC production in normal human epidermal keratinocytes (NHEK) in vitro. METHODS: the expression of TARC mRNA and protein were examined in NHEK and HaCaT cells stimulated with various cytokines. RESULTS: stimulation with inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha failed to induce TARC mRNA expression in NHEK. However, stimulation with IFN-gamma and TNF-alpha together enhanced expression slightly. ELISA analysis failed to detect TARC protein in NHEK culture supernatant, even following stimulation with IFN-gamma and TNF-alpha. In contrast, HaCaT cells produced TARC protein even without stimulation of cytokines. CONCLUSION: these results indicate that production of TARC by HaCaT cells is a phenomenon specific to the cell line and the observation on TARC in HaCaT cells can not be generalized. NHEK do not produce TARC protein in vitro.     

Identification of TIA-1+ and granzyme B+ cytotoxic T cells in lichen sclerosus et atrophicus

BACKGROUND: The onset and persistence of cutaneous lichen sclerosus et atrophicus (LSA) are linked to the presence of an inflammatory infiltrate of CD3+ T cells that includes CD4+ and CD8+ cells. The functional relevance of the presence of these cells is unknown. OBJECTIVE: The study intended to quantify resting and activated cytotoxic T cells in LSA lesions. METHODS: Twenty patients with active LSA were studied. Skin-infiltrating T cells were immunohistologically characterized with antibodies against CD3, CD8, T-cell-restricted intracellular antigen (TIA-1) and granzyme B (GrB). TIA-1 labels cytotoxic granules of resting and activated T cells, whereas GrB designates activated cytotoxic T lymphocytes (CTL). RESULTS: In all cases, numerous T cells were consistently found expressing cytotoxic granules. The results indicated a high number of infiltrating CD8+ TIA+ T cells. Furthermore, a notable number of GrB+ activated CTL associated with hydropic degeneration of the basal cell layer were found within the dermal infiltrate and at the dermoepidermal interface. CONCLUSION: This study shows that a high proportion of skin-infiltrating T cells in LSA has a potential cytotoxic function. The results indicate that hydropic degeneration of basal keratinocytes may at least partially be mediated by CTL-dependent mechanisms. Our data also indicate that a cell-mediated immune response may play an important role in the pathogenesis of the disease.     

Both IL-4 and IL-13 inhibit the TNF-alpha and IFN-gamma enhanced MDC production in a human keratinocyte cell line,HaCaT cells

BACKGROUND: Macrophage-derived chemokine (MDC) is a Th2 type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Recent reports demonstrated that MDC is expressed not only by macrophages, dendritic cells and lymphocytes, but also by cultured human keratinocytes (KCs). However, the regulation of MDC production in KCs by various cytokines has not been well documented. OBJECTIVE: In this study, we investigated how Th1/Th2 cytokines regulate MDC production in a human KC cell line, HaCaT cells. METHODS: HaCaT cells were cultured with or without various cytokines for 24 h and RT-PCR was performed using these cells to evaluate MDC mRNA levels. ELISA was carried out using supernatant of HaCaT cells to calculate secreted MDC protein levels. RESULTS: MDC mRNA was weakly expressed in HaCaT cells, and upon stimulation with TNF-alpha or IFN-gamma, MDC expression was strongly upregulated. The supernatant MDC levels when stimulated with TNF-alpha or IFN-gamma were significantly higher than those without stimulation, and were synergistically increased when stimulated with a combination of TNF-alpha and IFN-gamma. Both interleukin-4 (IL-4) and IL-13 inhibited TNF-alpha and IFN-gamma enhanced MDC production in HaCaT cells in a dose-dependent manner. CONCLUSION: Th2-type cytokines IL-4 and IL-13 downregulate the production of MDC, a Th2 type chemokine, by KCs. This may partially contribute to maintaining Th1/Th2 balance in inflammatory skin diseases like atopic dermatitis.     

Evaluation of the potential role of cytokines in toxic epidermal necrolysis

Toxic epidermal necrolysis is a rare disease observed as a consequence of adverse reactions to drugs. It results in the widespread apoptosis of epidermal cells and has a high mortality rate. The mechanisms leading to this apoptosis are not yet elucidated. We investigated whether the cytokines present in the blister fluid, which accumulates under necrotic epidermis, originated from T lymphocytes and may play a role in the propagation of keratinocyte apoptosis. Interferon gamma (IFN-gamma), soluble tumor necrosis factor alpha (TNF-alpha), soluble Fas ligand (sFas-L) were present in much higher concentration in the blister fluids of 13 toxic epidermal necrolysis (TEN) patients than in control fluids from burns. The results of RT-PCR studies, however, indicated that only IFN-gamma and to a lesser extent interleukin (IL)-18 were produced by mononuclear cells present in the fluid. That suggests that the other cytokines also present (TNF-alpha, sFas-L, IL-10) rather originated from activated keratinocytes. Fas-L was indeed overexpressed on the membranes of keratinocytes in lesional skin in situ. The Th1 profile of T lymphocyte activation found in the blister fluid of patients with TEN is consistent with a key role for drug-specific cytotoxic T lymphocytes (CTL) as previously reported, the activation of keratinocytes by IFN-gamma making them sensitive to cell-mediated cytolysis. We propose the hypothesis that the production of Fas-L, TNF-alpha, and IL-10 by keratinocytes could be a defense mechanism against CTL rather than a way of propagating apoptosis among epidermal cells.     

The activity of caspase-1 is increased in lesional psoriatic epidermis

Caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the proinflammatory cytokine IL-18, a cytokine known to play an important role in the pathogenesis of psoriasis. The purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress-induced activation of caspase-1 and IL-18. Interestingly, increased caspase-1 activity in lesional compared with non-lesional psoriatic skin was seen. In vitro experiments in cultured human keratinocytes demonstrated anisomycin-induced, p38 mitogen-activated protein kinase (p38 MAPK)-dependent increased secretion of procaspase-1 and active caspase-1. Furthermore, anisomycin increased the mRNA expression of IL-18 through a p38 MAPK-dependent but caspase-1-independent mechanism, reaching a maximum level after 12 hours of stimulation. Finally, anisomycin caused a rapid (4 hours) increase in the secretion of proIL-18 and active IL-18. Secretion of active IL-18 was mediated through a p38 MAPK/caspase-1-dependent mechanism, whereas secretion of proIL-18 was mediated by a p38 MAPK-dependent but caspase-1-independent mechanism. These data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that IL-18 secretion is regulated by a p38 MAPK/caspase-1-dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis.     

Expression of wild-type, but not mutant, loricrin causes programmed cell death in HaCaT keratinocytes

The epidermal cornified cell envelope is a complex protein-lipid composite that replaces the plasma membrane of corneocytes and is crucial for epidermal barrier function. Loricrin is a major constituent of the epidermal cornified cell envelope, contributing approximately 70% by mass. In order to explore novel function of wild-type (WT) loricrin other than the major component of the epidermal cornified cell envelope, we transiently expressed construct encoding human WT and mutant loricrin (730insG) in HaCaT keratinocytes. HaCaT cells transfected with WT or mutant loricrin were at differentiation level. WT loricrin in the transfected cells was seen diffusely in the cytoplasm and nuclei. Positive transferase deoxytidyl uridine end labeling staining was observed in the nuclei of WT loricrin-transfected HaCaT keratinocytes. Data from the DNA fragmentation assay showed that only WT loricrin induced DNA ladders compared with that of mutant loricrin. WT loricrin-transfected HaCaT keratinocytes were susceptible to programmed cell death (PCD). Activation of caspase-14 was also seen. In contrast, PCD or activation of caspase-14 did not occur in mutant loricrin-transfected HaCaT cells. These results suggest that the expression of WT loricrin facilitates induction of PCD in HaCaT keratinocytes.     

Differences in responses of interleukin-1 and tumor necrosis factor α production and secretion to cyclosporin-A and ultraviolet B-irradiation by normal and transformed keratinocyte cultures

Abstract Among epidermal cytokines, IL-1 and TNFα are involved in inflammatory skin reactions and suspected of modulation by immuno-suppressive treatment (e.g., cyclosporin A, CsA) or UVB-irradiation, 2 mediators probably being involved in epithelial carcinogenesis. We evaluated the effects of 8 μg/ml CsA and 100 J/m² UVB-irradiation on the production and secretion of IL-1 and TNFα on normal human epidermal keratinocytes (NHK) and epidermal keratinocyte cell lines either spontaneously transformed (HaCaT) or transformed by human papillomavirus (HPV) type 16 or 18 (EK I6 and EK 18), by using FLISA test. Normal and immortalized keratinocytes constitutively produced and released IL-lα IL-lβ and IL-1 receptor antagonist (IL-IRA) but IL-I synthesis by NHK was significantly higher than by cell lines. All the cells spontaneously excreted low amounts of TNFα. Different responses to treatments were evidenced between NHK and cell lines. CsA modified significantly the production and secretion of ILI in most cells whereas slight changes were observed with TNFα secretion. UVB irradiation had no effect on the intracellular ILI pool of any cells but increased the release of IL1 and TNFα. The association CsA-UVB did not result in additive effects on synthesis and secretion of IL1; the release of TNFα by the cells remained poor except for EK18 cells. Taken together, these results show that, in immortalized keratinocytes, the IL-1 and TNFα expression was differently affected by treatments wilh CsA and/or UVB-irradiation as compared to NHK. In addition, spontaneously transformed keratinocytcs. HaCaT, reacted differently from HPV-transformed keratinocyles, EK I6 and EK I8.     

银屑病患者真皮间充质干细胞对HaCaT细胞的作用

目的 研究分析不同浓度银屑病患者真皮间充质干细胞（Dennis mesenchymal stem cells,DMSCs）对人永生化角质形成细胞（HaCaT）的作用.方法 取银屑病患者和正常人的真皮,获得纯度高的DMSCs,将HaCaT在Transwell小室下室接种,同时按照第5代DMSCs与HaCaT细胞之间的比例（1：1、5：1、10：1）将DMSCs分3组接种在Transwell上室,并设正常对照组和自然增殖组,采用实时细胞分析系统（RTCA）对HaCaT细胞进行增殖检测,培养74 h后用细胞计数法检测HaCaT细胞的数量.结果 共培养组较自然增殖组HaCaT细胞计数减少,与健康对照组相比,银屑病患者组HaCaT细胞计数减少不明显,以银屑病患者DMSC与HaCaT细胞按照1：1共培养组为著.结论 ①DMSCs对HaCaT细胞增殖有抑制作用.②与正常对照组、自然增殖组和其他比例组相比银屑病患者DMSCs与HaCaT 1：1共培养组对HaCaT细胞增殖抑制作用减弱更显著.     

Characterization of a T cell line bearing natural killer receptors and capable of creating psoriasis in a SCID mouse model system

T cells bearing natural killer receptors (NKRs) such as CD94 and CD161 are present in psoriasis. These immunocytes express several receptors for both classical and non-classical class I MHC molecules. Whether T cells bearing NKRs in psoriatic lesions represent immunoregulatory versus pathogenic immunocytes or are just bystander cells is unclear. To address this issue, a CD94+/CD161+ T cell line was established from a psoriatic patient using IL-2/superantigens, and the interaction between NK-T cells and keratinocytes was characterized using in-vitro and in-vivo assays. Upon incubation between NK-T cells and CD1d positive keratinocytes, high levels of IFN-gamma and IL-13 were produced. Cytokine production was inhibited by a mAb against CD1d, implicating recognition of this surface molecule in the T cell response. Furthermore when this line was injected into pre-psoriatic skin engrafted onto a SCID mouse, a psoriatic plaque was created. The hyperplastic epidermal keratinocytes diffusely expressed CD1d, and were infiltrated by CD161+ T cells. RNase protection assay revealed predominantly IFN-gamma and IL-15 mRNAs, with barely detectable IL-13 mRNA in the acute lesion. These in-vivo findings demonstrated that this T cell line was pathogenic by creating a psoriatic plaque. The in-vitro results support a pathophysiologic role for interaction between T cells expressing NKRs and CD1d positive keratinocytes, with subsequent production of IFN-gamma. Upon injection in-vivo, the cytokine network produced was characterized by an immunological response polarized towards Th1 rather than Th2 cytokines. Thus, this pathogenic cell line provides evidence that T cells bearing NKRs can directly provoke a Th1 disease such as psoriasis.     

Interleukin‐1 from epithelial cells fosters T cell‐dependent skin inflammation

Background Chronic inflammatory skin diseases such as atopic dermatitis and psoriasis are characterized by the infiltration of lymphocytes into the epidermal compartment. Several studies point to an active role of skin epithelial cells in the pathophysiology of such diseases. Objectives In this study we addressed the regulatory function of primary human keratinocytes in the interaction with autologous T cells and monocytes. Methods We used a human coculture model with keratinocytes grown from epidermal stem cells of the outer root sheath of human hair follicles and autologous T cells. Results In our coculture system we observed a high production of interferon (IFN)‐γ, but not Th2 cytokines, in the presence of superantigen or antigen‐pulsed autologous monocytes. Critical parameters for this effect were: (i) T‐cell receptor activation, (ii) an intercellular adhesion molecule‐1/lymphocyte function‐associated antigen (LFA)‐1‐dependent interaction between keratinocytes and T cells, and (iii) secretion of interleukin (IL)‐1β. Remarkably, in the presence of activated T cells, epithelial cells seemed to be a more significant source of IL‐1β than monocytes. Application of the LFA‐1 blocker efalizumab or IL‐1 receptor antagonist anakinra enabled us to suppress completely the production of IFN‐γ by T cells in the coculture. Conclusions IL‐1 secretion and the physical contact between keratinocytes and activated, infiltrating T cells may be central for the development of chronic inflammatory skin conditions.     

Influence of psoriatic peripheral blood CD4+ T and CD8+ T lymphocytes on C-myc, Bcl-xL and Ki67 gene expression in keratinocytes

T cells play a role in the hyperproliferation of keratinocytes, however, direct evidence on what is the main mechanism and which T-cell subset is central is still lacking. Our aim was to elucidate the role and mechanism of CD4+T and CD8+ T lymphocytes in the immunopathogenesis of lesion hyper-proliferation in psoriasis. T cell and CD4+T and CD8+ T cell subpopulations were isolated by immunomagnetic methods from peripheral blood of psoriatic patients and healthy volunteers. Keratinocytes from foreskins were incubated in the absence or presence of T cells and T cell subpopulations for 72 h. The expression of c-Myc, Bcl-xL and Ki67 proteins in keratinocytes were detected by immunohistochemical staining, and then IL-4, IL-8 and IFNgamma level in the supernatant was determined by respective ELISA. T cells of psoriatic origin induced keratinocytes to overexpress C-myc and Ki67 proteins with high amounts of IL-8 and IFN-gamma in the supernatant, while the T cells of normal origin did not. Furthermore, there was a difference in keratinocyte response to CD4+ T cells and CD8+ T cells from psoriatic patients. CD4+ T cells can secrete much higher levels of IL-8 and IFN-gamma and induced much stronger C-myc and Ki67 expression in keratinocytes, compared with CD8+ T cells. We concluded that psoriatic peripheral blood T cells and the T cell subpopulation could induce keratinocytes to over-express pro-proliferation proteins probably by secreting Th1 cytokines. T cells, especially the CD4+ T cell subpopulation, play an important role in the immunopathogenesis of lesion hyper-proliferation in psoriasis.     

阿维A对人表皮鳞状细胞癌A431细胞株生长及凋亡的影响

目的 研究阿维A对上皮鳞状细胞癌(鳞癌)细胞生长的影响及其诱导凋亡作用。方法 以体外培养的人表皮鳞癌细胞A431细胞和角质形成细胞HaCaT细胞为研究对象,用MTT法观察阿维A对A431细胞和HaCaT细胞的生长抑制作用,用光镜和电镜观察细胞形态,流式细胞仪检测凋亡峰的出现,Annexin-V染色证实凋亡的发生。结果 随着阿维A作用时间的延长及剂量的增加,对A431细胞增殖的抑制作用增加。同样浓度和同样作用时间的阿维A对A431细胞增殖的抑制率高于对HaCaT细胞的抑制率。光镜和电镜观察显示随阿维A作用时间延长,A431凋亡细胞增多。流式细胞仪检测显示随阿维A作用时间的延长,亚G1期凋亡峰明显增加,Annexin-V染色阳性细胞数增多。结论 阿维A具有抗上皮鳞癌细胞生长及诱导上皮鳞癌细胞凋亡作用。     

DNA-dependent protein kinase catalytic subunit is cleaved during UV-induced apoptosis

Ultraviolet radiation can induce the injury of epidermal keratinocytes, resulting in sunburn cell (apoptotic cell) formation. It has been demonstrated that the protease caspase-3, a downstream molecule of the CD95 pathway, is activated in UV-exposed HaCaT cells, and that the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is cleaved by interleukin-1beta converting enzyme (ICE)-like protease during apoptosis induced by X-rays, staurosporine and etoposide. Then, we studied whether the DNA-PKcs is cleaved during UV-induced apoptosis in keratinocytes. We used the well-characterized cloned human keratinocyte cell line HaCaT, which carries p53 mutations. UVB-induced apoptotic cells were observed by TdT-mediated deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay and agarose gel electrophoresis. Western blot analysis was performed using the antibody against DNA-PKcs. The cleavage occurred during UVB-induced apoptosis in HaCaT cells. It suggests that the cleavage is associated with loss of DNA-PK activity. Thus, a functional relevance of cleavage of DNA-PKcs may be to prevent rejoining fragmented DNA during apoptosis, thereby promoting apoptotic processes. Although apoptosis was not completely blocked by the caspase-3 inhibitor, the cleavage of the DNA-PKcs was blocked. These results indicate that DNA-PKcs is cleaved by the caspase-3 for UVB-induced apoptosis in HaCaT cells.     

Thymus and activation regulated chemokine (TARC)/CCL17 and skin diseases

Thymus and activation-regulated chemokine (TARC)/CCL17 is constitutively expressed in the thymus and is produced by dendritic cells (DC), endothelial cells, keratinocytes (KC) and fibroblasts. TARC is designated a Th2 type chemokine since it binds to CCR4. We review the pathogenic role of TARC in skin diseases such as atopic dermatitis (AD), bullous pemphigoid (BP) and mycosis fungoides (MF) focusing on epidermal KC and Langerhans cells (LC), which are epidermal DC. We have determined that serum TARC levels sharply reflect the disease activity of AD, which is thought to be a Th2-dominant inflammatory skin disease especially in the acute phase. Serum TARC levels are also related to the disease activity of BP, which is a blistering autoimmune skin disease, and MF, which is a cutaneous T-cell lymphoma, but very high serum TARC levels are only seen in a limited number of various other skin diseases. TARC may be a useful laboratory marker for the diagnosis of AD, especially cases which are moderate to severe, and for the evaluation of disease activity of AD. IL-4 and TGF-beta1 downregulate TNF-alpha and IFN-gamma induced TARC production in the human KC cell line, HaCaT cells, while IL-4 upregulates, and IFN-gamma downregulates TARC production by mouse LC. Because TARC and its receptor CCR4 are believed to play important roles in the pathogenesis of AD, BP and MF, TARC and CCR4 may be possible future targets for therapy of these diseases.     

Interleukin-1β upregulates tissue-type plasminogen activator in a keratinocyte cell line (HaCaT)

Human keratinocytes synthesize and secrete tissue-type plasminogen activator (tPA). tPA converts the inactive precursor enzyme plasminogen into the trypsin-like proteinase plasmin. tPA is not found in normal epidermis, but in lesional epidermis from patients with a variety of cutaneous diseases, including psoriasis, pemphigus and pemphigoid. The presence of tPA is probably a reaction to the disease process rather than the initiating event in these etiologically and histopathologically diverse lesions. However, the factor(s) that upregulate tPA expression and secretion in keratinocytes have remained largely elusive. We sought to determine whether the inflammatory cytokine interleukin-1β (IL-1β), which is commonly present in diverse epidermal lesions, influences tPA production. Accordingly, we studied the influence of IL-1β on secretion of tPA by cells of the human keratinocyte cell line HaCaT. We found that IL-1β increased tPA secretion in these cells. Given the observation that IL-1β is a common proinflammatory mediator in cutaneous diseases, our findings may explain the increase in tPA in clinically and etiologically diverse inflammatory epidermal lesions. Received: 9 October 1995     

Effect of Spa Spring Water on Cytokine Expression in Human Keratinocyte HaCaT Cells and on Differentiation of CD4+ T Cells

Background

Skin acts as the first line of defense against any foreign materials outside of our body. In inflammatory skin disease, the pathogenesis is due to an immune reaction in the keratinocytes, immune cells and soluble mediators. Balneotherapy is widely used for the treatment of inflammatory skin disease, but the mechanisms are only partly understood by immune regulation. Balneotherapy in dermatologic disease can affect the secretion of pro-inflammatory cytokines, IL-1α and tumor necrosis factor from keratinocytes, and possibly affect the T cell differentiation.

Objective

In this study, we evaluated the effect of spa spring water from Yong-gung oncheon on the cells, and investigated the skin immune reaction.

Methods

We investigated the immunomodulatory or anti-inflammatory effect of thermal spring water on the expression of pro-inflammatory cytokines in the HaCaT cells under Toll-like receptor (TLR) stimulation, as well as the effect on the differentiation of CD4⁺ T cells under spring water.

Results

The treatment of spa spring water from Yong-gung oncheon decreased the expression of proinflammatory cytokines under TLR stimulation to the HaCaT cells and antigen presenting cells. In addition, spa spring water attenuated the differentiation process of subsets of CD4⁺ T cells, i.e., Th1, Th2 and Th17 cells. All these immune parameters can be used to evaluate the efficacy of spa spring water in Korea, in terms of the immune modulatory effect.

Conclusion

Spa spring water treatment suppressed the inflammatory cytokines production and also modulated the differentiation of CD4⁺ T cells into Th1, Th2, and Th17 cells, but not the T_regs cells.     

Interleukin-7. Biology and implications for dermatology

Abstract In recent years, it has become apparent that IL-7, originally characterized as a growth factor for pre-B lymphocytes. also has important implications for the skin. Keratinocytes have been shown to produce IL-7, which in turn can elicit a variety of biological responses on several cell types residing in the skin. IL-7 has been demonstrated to augment the cytolytic activity of cytotoxic T cells (CTL) and natural killer (NK) cells against various neoplastic targets indcluding melanoma cells. Proliferation and long-term survival of murine dendritic epidermal T lymphocytes (DETC) in vitro is supported by IL-7. IL-7 also induces secretion of inflammatory cytokines by monocytes/macrophages and renders these cells to become tumoricidal against melanoma cells. Normal and malignant melanocytes respond to IL-7 with increased expression of intercellular adhesion molecule (ICAM-I). In addition, IL-7 has been shown to act as growth factor for Sczary cells, suggesting a röle of keratinocyte-derived IL-7 in the pathogenesis of cutaneous T cell lymphoma. Because of the potent in vitro immunomodulatory effects of IL-7 which have been confirmed in mouse tumor models, IL-7 may become a valuable additional agent to immunotherapeutical regimens currently studied in patients with advanced melanoma. This review summarizes our present knowledge about the molecular and immunological properties of IL7 with emphasis on the effects of that cytokine within the cutaneous compartment and the potentical clinical utility in dermatology.