Proliferation and interferon-γ receptor expression in psoriatic and healthy keratinocytes are influenced by interactions between keratinocytes and fibroblasts in a skin equivalent model

Epidermal-dermal interactions were studied in a skin equivalent model. Six combinations of keratinocytes and fibroblasts from healthy and psoriatic skin were used. TPA (12-O-tetradecanoylphorbol-13-acetate) was used to determine whether the expression of the IFN- receptors in keratinocytes was related to epidermal differentiation and proliferation. These phenomena were assessed by immunohistochemistry. In all epidermal outgrowths, the epidermal growth factor receptor was expressed throughout the epidermis, cytokeratin 16 suprabasally, and filaggrin and involucrin in its superficial part. The IFN- receptor was expressed throughout the epidermis, but was unevenly distributed. The expression of the IFN- receptor was quantified by confocal laser scanning microscopy both in the whole of epidermis and in areas with the strongest intensity. The total amount varied to a minor degree in the epidermal outgrowths of different origins and was unaffected by TPA. In high-intensity areas interactions between keratinocytes and fibroblasts did influence the amount of IFN- receptor expression and TPA decreased the expression by 13%. There was no correlation between the proliferation rate and the expression of the IFN- receptor. Psoriatic and healthy keratinocytes were equally well differentiated in the skin equivalents. The interferon- receptor was similarly expressed under these conditions. The growth rate, assessed by Ki-67-positive nuclei in the basal layer, was highest in healthy keratinocytes. Keratinocytes from psoriatic lesions increased their growth rate when cocultured with psoriatic fibroblasts compared with normal ones, indicating that fibroblasts may be of importance for epidermal hyperproliferation in psoriatic lesions.     

RXRα ablation in epidermal keratinocytes enhances UVR-induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes

We show here that keratinocytic nuclear receptor retinoid X receptor-α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute UVR. Keratinocytic RXRα has a protective role in UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress-mediated DNA damage, and cellular apoptosis. We discovered that keratinocytic RXRα, in a cell-autonomous manner, regulates mitogenic growth responses in skin epidermis through secretion of heparin-binding EGF-like growth factor, GM-CSF, IL-1α, and cyclooxygenase-2 and activation of mitogen-activated protein kinase pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as endothelin 1, hepatocyte growth factor, α-melanocyte stimulating hormone, stem cell factor, and fibroblast growth factor-2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRα(ep-/-) mice), which in a non-cell-autonomous manner modulated melanocyte proliferation and activation after UVR. RXRα(ep-/-) mice represent a unique animal model in which UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, the results of our study suggest that RXR antagonists, together with inhibitors of cell proliferation, can be effective in preventing solar UVR-induced photocarcinogenesis.     

Fcγ receptors on keratinocytes in psoriasis

Summary Epidermis in cryostat sections of skin biopsy specimens from patients with psoriasis and from healthy individuals bound bovine erythrocytes (E) sensitized with rabbit IgG antibodies (A)(EA). No binding occurred using E or E sensitized with IgM or F(ab)₂ fragments of IgG. The binding of EA was inhibited by human IgG and by Fc fragments of IgG, whereas human IgA, IgM, albumin, and F(ab)₂ fragments of IgG did not inhibit the binding, indicating the presence of receptors for the Fc part of IgG (FcR).EA bound mainly to stratum spinosum and most strongly above FcR-positive cell infiltrates in dermal papillae. The binding of EA to sections from patients with active psoriasis was stronger than to sections from patients with stationary psoriasis vulgaris. Sections of unaffected skin from patients with psoriasis and healthy individuals also bound EA, but the binding was weaker than to sections of psoriatic lesions. The receptors were sensitive to periodic acid, formaldehyde, and heat.Using immune complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP, the receptors were localized to the outer aspect of the keratinocytes and to the inflammatory cells in the microabscesses. The strongest binding occurred in the same areas which adhered EA most strongly. FcR on dendritic epidermal cells could not be demonstrated in situ. A monoclonal antibody against FcR also stained the outer aspect of most keratinocytes throughout the epidermis.FcR on keratinocytes support the assumption that these cells contribute to immune reactions in the skin.     

Effects of interferon-γ treatment on the cutaneous DTH reaction in rats

Summary The capacity of interferon- (IFN-) to induce class II histocompatibility antigens on different cell types including keratinocytes, is well known, but the impact of IFN- on the immune response is still unclear. Lewis rats sensitized with dinitrofluorobenzene (DNFB) were injected with recombinant rat IFN- (10⁵ U) or phosphate-buffered saline (PBS) once daily on 3 successive days at the bases of the ears either before or after they were challenged on the ears. As expected, the PBS-treated animals showed about a 30% increase in ear thickness and there was an induced expression of class II antigens on the keratinocytes as judged by immunohistochemistry 72 h after challenge. Exogenously added IFN- prior to DNFB challenge resulted in a significantly reduced ear swelling at 24 (p<0.01) and 48 h (p<0.05) after challenge. In this case the keratinocytes expressed class II antigens already at the time of challenge. When IFN- injections were given during the contact allergic reaction there was no significant reduction of ear swelling until 72 h (p<0.01). At that time point there was a more pronounced expression of class II antigens on the keratinocytes compared with PBS-injected animals, due to the IFN- treatment. These in vivo data support our previous observations that IFN- may play a self-limiting role in certain immune responses.     

Induction of mRNA for HLA-DRβ in human keratinocytes cocultured with interferon-gamma

Summary Explanted human keratinocytes exposed in vitro to recombinant interferon-gamma (IFN-) were investigated for the appearance of mRNA for HLA-DR. Using in situ hybridization with a (³⁵S)UTP-labelled HLA-DR cRNA probe, mRNA-positive cells were detected already within 6 h with maximal numbers of positive cells as well as the amount of mRNA per cell after 48 h. The corresponding protein HLA-DR, as analysed by immunoperoxidase staining, was detected on 20%–40% of the cells after 24 h and on almost all cells within 48 h. The expression of HLA-DQ and-DP antigens were always exceeded by that of HLA-DR. Whereas an increase in the concentration of IFN- above 50 U/ml did not affect the maximal level of HLA-DR reactive cells, there was a fourfold increase in the frequency of cells reactive with HLA-DQ and a twofold increase for HLA-DP when the IFN- concentration was raised from 50 to 500 U/ml. When IFN- was withdrawn from the cultures, HLA-DR mRNA and protein synthesis ceased — indicating the continuous need for IFN- to maintain the HLA-DR synthesis in keratinocytes.     

Fas-FasL interaction in cytotoxic T cell-mediated vitiligo: The role of lesional expression of tumor necrosis factor-α and interferon-γ in Fas-mediated melanocyte apoptosis

Vitiligo is an acquired skin depigmentation disorder resulting from the selective loss of epidermal melanocytes, and previous studies have suggested that a T lymphocyte-mediated mechanism has a role in melanocyte loss. Although Fas-Fas ligand (FasL) interactions are important for T lymphocytes to mediate cytotoxicity, there are only few reports examining the involvement of the Fas-FasL pathway in vitiligo using in vivo mouse models. In addition, there have been no reports concerning Fas-mediated apoptosis in human melanocytes in vitro. In this study, we found that the Fas-mediated pathway is involved in cytotoxic T lymphocyte (CTL)-dependent vitiligo in a mouse model and FasL-induced apoptosis of human melanocytes. Tumor necrosis factor (TNF)-α and interferon (IFN)-γ, the expression levels of which have been reported to be elevated in lesional skin of patients with vitiligo, synergistically upregulated Fas expression on human melanocytes but inhibited the Fas-mediated apoptosis. Treatment with TNF-α and IFN-γ synergistically upregulated the expression of the anti-apoptotic genes, c-IAP2, c-FLIP and MCL1. A siRNA knock-down study showed that c-FLIP and MCL1, but not c-IAP2, were involved in inducing synergistic inhibitory effects on Fas-mediated apoptosis. Furthermore, we found that FasL and TNF-related apoptosis - inducing ligand (TRAIL) synergistically induced apoptosis on human melanocytes. In conclusion, our results suggest that the Fas-FasL pathway is involved in CTL-dependent vitiligo and the elevated expression levels of TNF-α and IFN-γ in lesional skin may act synergistically on melanocytes to suppress Fas-mediated apoptosis.     

Human keratinocytes express AIM2 and respond to dsDNA with IL-1β secretion

Keratinocytes have been recognized to actively participate in the skin immune response. It has been shown that keratinocytes express all components that are necessary to form the NLRP3 inflammasome complex including the adapter protein ASC and caspase-1. In this study, we investigated the presence and activity of the recently identified absent in melanoma 2 (AIM2) inflammasome in human keratinocytes. We were able to show that an AIM2 inflammasome is active in human keratinocytes. IL-1 production by keratinocytes plays a pivotal role in inflammatory processes in the skin. Activation of the AIM2 inflammasome in keratinocytes represents another potential trigger factor for the development and maintenance of inflammatory skin diseases.     

Effect of α-lipoic acid on oxidative stress of keratinocytes induced by UVB

Objective;To determine the effect of α-lipoie acid on human primary keratinocyte oxidative stress induced by UVB radiation. Methods;The oxidative stress model was established by H2O2and UVB. The cell morphology and fluorescent intensity were observed under inverted microscope or fluorescence microscope. The intracellular ROS was detcted with fluorescent nuclear staining of 2'7' - acetyl chloride fluorescence sodium acetate (DCFH-DA). Green fluorescent was quantified by flow cytometry. The total antioxidant capacity of total antioxidant capacity (TAOC) and malonaldehyde (MDA) were measured by chemical method. Results;The fluorescence intensity and MDA in cells irradiated by UVB for 4hs were 35311±3072.3 and 11.61 ±0.68 nmol/mg · prot, which were higher than that in negative control group (24465 ±2085.3 and 8.25± 1.21), with significant differences (Ps<0.05). After incubated 4hs with antioxidant LA, the fluorescence intensity and MDA were reduced to 26926±3502.7 and 9.14+1.12 vnmol/mg · prot. There was no significant difference in the level of TAOC between different groups. Conclusion;LA can alleviate the oxidative stress of the keratinocyte induced by UVB irradiation. © 2019 Shandong Yinbao Technology Co. Ltd. All rights reserved.     

A single intradermal injection of IFN-γ induces an inflammatory state in both non-lesional psoriatic and healthy skin

Psoriasis is a chronic, debilitating, immune-mediated inflammatory skin disease. As IFN-γ is involved in many cellular processes, including activation of dendritic cells (DCs), antigen processing and presentation, cell adhesion and trafficking, and cytokine and chemokine production, IFN-γ-producing Th1 cells were proposed to be integral to the pathogenesis of psoriasis. Recently, IFN-γ was shown to enhance IL-23 and IL-1 production by DCs and subsequently induce Th17 cells, which are important contributors to the inflammatory cascade in psoriatic lesions. To determine whether IFN-γ indeed induces the pathways expressed in psoriatic lesions, a single intradermal injection of IFN-γ was administered to an area of clinically normal, non-lesional (NL) skin of psoriasis patients and biopsies were collected 24 hours later. Although there were no visible changes in the skin, IFN-γ induced many molecular and histological features characteristic of psoriatic lesions. IFN-γ increased a number of differentially expressed genes in the skin, including many chemokines concomitant with an influx of T cells and inflammatory DCs. Furthermore, inflammatory DC products tumor necrosis factor (TNF), inducible nitric oxide synthase, IL-23, and TNF-related apoptosis-inducing ligand were present in IFN-γ-treated skin. Thus, IFN-γ, which is significantly elevated in NL skin compared with healthy skin, appears to be a key pathogenic cytokine that can induce many features of the inflammatory cascade of psoriasis.