Transcriptional regulation of the MAIL gene in LPS-stimulated RAW264 mouse macrophages

IkappaB inhibits nuclear factor kappa B (NF-kappaB), which is known to regulate the expression of various genes, including genes involved in inflammation. Recently, a novel IkappaB family protein, 'molecule possessing ankyrin repeats induced by lipopolysaccharide' (MAIL), was identified. MAIL is a nuclear-acting, inducible protein, unlike typical IkappaB proteins. However, the mechanism of its induction by lipopolysaccharide (LPS) is unclear. Using the LPS-reactive region located upstream from the MAIL gene, we investigated the mechanism of MAIL induction. MAIL expression was strongly regulated by NF-kappaB and partly regulated by CREB. Furthermore, deletion, point mutation and binding analyses revealed that the NF-kappaB binding site located at -229 to -220 bp is an essential target of MAIL expression. Overexpression of MAIL protein suppressed the LPS-induced promoter activity of the MAIL gene. These data indicate that MAIL expression is strongly upregulated by NF-kappaB, and it is controlled, at least in part, by an autoregulation mechanism.     

A second step of chemotaxis after transendothelial migration: keratinocytes undergoing apoptosis release IFN-gamma-inducible protein 10, monokine induced by IFN-gamma,and IFN-gamma-inducible alpha-chemoattractant for T cell chemotaxis toward epidermis in atopic dermatitis

Activation and skin-selective homing of T cells and their effector functions in the skin represent sequential immunological events in the pathogenesis of atopic dermatitis (AD). Apoptosis of keratinocytes, induced mainly by T cells and mediated by IFN-gamma and Fas, is the essential pathogenetic event in eczema formation. Keratinocyte apoptosis appears as activation-induced cell death in AD. By IFN-gamma stimulation, chemokines such as IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and IFN-gamma-inducible alpha-chemoattractant are strongly up-regulated in keratinocytes. These chemokines attract T cells bearing the specific receptor CXCR3, which is highly expressed on T cells isolated from skin biopsies of AD patients. Accordingly, an increased T cell chemotaxis was observed toward IFN-gamma-treated keratinocytes. Supporting these findings, enhanced IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and IFN-gamma-inducible alpha-chemoattractant expression was observed in lesional AD skin by immunohistochemical staining. These results indicate a second step of chemotaxis inside the skin after transendothelial migration of the inflammatory cells. Keratinocytes undergoing apoptosis in acute eczematous lesions release chemokines that attract more T cells toward the epidermis, which may further augment the inflammation and keratinocyte apoptosis.     

Selective I kappa B kinase expression in airway epithelium generates neutrophilic lung inflammation

To determine whether NF-kappaB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IkappaB kinase 1 (cIKK1) or IkappaB kinase 2 (cIKK2) in airway epithelium. After intratracheal administration of adenoviral vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that express Photinus luciferase under the control of an NF-kappaB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing beta-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-kappaB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-kappaB activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated mice was confirmed by immunoblots for RelA and EMSA from lung nuclear protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed induction of mRNA expression of several chemokines and cytokines in lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or Ad-cIKK2 showed elevated concentrations of NF-kappaB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-kappaB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-kappaB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-kappaB activation by these kinases. These data show that selective expression of IkappaB kinases in airway epithelium results in NF-kappaB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-kappaB in lung epithelium may be beneficial in treating inflammatory lung diseases.     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.     

Reactive nitrogen species formation in eosinophils and imbalance in nitric oxide metabolism are involved in atopic dermatitis-like skin lesions in NC/Nga mice

Nitric oxide (NO) and reactive nitrogen species (RNS) have been implicated in the pathogenesis of inflammatory diseases. However, the involvement of NO and RNS in atopic dermatitis (AD), a pruritic inflammatory skin diseases, is not fully understood. In this study, we investigated the contribution of NO and RNS to the development of AD-like skin lesions in NC/Nga mice, an animal model for human AD. AD-like skin lesions were observed in NC/Nga mice kept under conventional conditions but not in specific pathogen-free conditions. The expression of inducible NO synthase (iNOS) and endothelial NOS (eNOS) proteins was upregulated in the dermal lesions, and that of neuronal NOS (nNOS) was downregulated in the epidermal lesions of the skin. Although the concentrations of NO2(-) and NO3(-) were lower, protein-bound nitrotyrosine content was significantly increased in the skin lesions. Immunohistochemical localization of nitrotyrosine was observed in almost all eosinophils. These results suggest that RNS formation in eosinophils and imbalance of NO metabolism are involved in the pathogenesis of AD-like skin lesions in NC/Nga mice.     

Inflammatory cytokine‐mediated induction of serine racemase in atopic dermatitis

Serine racemase (SR) is an enzyme that catalyses the synthesis of d ‐serine, an endogenous coagonist for N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild‐type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune‐reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d ‐serine in stratum corneum in AD patients and healthy controls using a tape‐stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d ‐serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor ‐α or macrophage migration inhibitory factor. Taken together, these findings suggest that d ‐serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.     

Rosa davurica Pall. improves DNCB-induced atopic dermatitis in mice and regulated TNF-Alpa/IFN-gamma-induced skin inflammatory responses in HaCaT cells

PurposeRosa davurica Pall., is mainly distributed in Korea, Japan, northeastern China, southeastern Siberia, and eastern Asia. It has been extensively used to treat various kinds of diseases by reason of the significant antioxidant, antiviral and anti-inflammatory activities. However, the pharmacological mechanism of Rosa davurica Pall. in atopic dermatitis (AD) is still ill defined and poorly understood. This study was to examine the anti-inflammatory effects and its mechanism on AD of Rosa davurica Pall. leaves (RDL).MethodsTo evaluate the therapeutic potential of RDL against AD, we have investigated the effects of RDL on the inflammatory reactions and the productions of inflammatory chemokines and cytokines that were induced by tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) in HaCaT cells. Futhermore, we examined the effects of RDL on the signaling pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB). For the in-vivo studies, RDL extract was topically applied to the dinitrochlorobenzene (DNCB)-induced AD mice, then its therapeutic effect was evaluated physiologically and morphologically.ResultsAfter the stimulation of HaCaT cells with TNF-α/IFN-γ, RDL considerably reduced the release of inflammatory mediators such as nitric oxide (NO), PEG₂ and other cytokines. RDL also reduced the phosphorylations of MAPK and NF-κB in TNF-α/IFN-γ-stimulated HaCaT cells. In vivo topical application of RDL to DNCB-induced AD mice significantly reduced the dorsal skin and ear thickness, clinical dermatitis severity, and mast cells. Treatment with RDL also markedly decreased the levels of serum IgE, IL-6 and the number of WBCs in the blood.ConclusionOur studies indicate that RDL inhibits the AD-like skin lesions by modulating skin inflammation. Consequently, these results suggest that RDL may be served as a possible alternative therapeutic treatment for skin disorder such as AD.     

Effect of nodakenin on atopic dermatitis-like skin lesions

Nodakenin, derived from the roots of Angelica gigas Nakai, is an important natural resource and medicinal material with anti-allergic and anti- inflammatory activities. We have previously shown that nodakenin inhibits IgE/Ag-induced degranulation in mast cells. In this study, we investigated the inhibitory effect of nodakenin on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)- like skin lesions in ICR mice. Scratching behavior, skin severity score, blood IgE level, and skin thickness were improved in DNCB-induced AD-like ICR mice. Our results showed that nodakenin suppressed the increase of AD-like skin lesions in ICR mice. These results suggest that nodakenin may be a potential therapeutic resource for AD as well as an adjunctive agent to control itching associated with AD.     

Development of atopic dermatitis-like skin lesions in STAT6-deficient NC/Nga mice

Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by elevation of plasma levels of total IgE, infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 T cells. However, the role of Th2 cells in the pathogenesis of AD is not fully understood. In this study we examined the NC/Nga (NC) mouse model of AD and established STAT6-deficient (SATA6(-/-)) NC mice to investigate the relevance of IL-4-mediated immune responses. Surprisingly, these mice elicited AD-like skin lesions at equivalent frequency and time of onset compared with normal NC littermates. Histological features of the lesion in STAT6(-/-) NC mice fulfilled the criteria for the pathogenesis of AD, although these mice fail to produce IgE and Th2 cytokines. The lymph nodes proximal to the regions of skin that developed lesions exhibited massive enlargement elicited by the accumulation of activated IFN-gamma-secreting T cells. Moreover, caspase I, IL-18, IL-12, and IFN-gamma are found to be highly expressed at the skin lesion, occurring simultaneously with elevation of eotaxin 2 and CCR3 expression. Therefore, the Th2-mediated immune response is not necessary for the development of AD-like skin disease in NC mice. The skin microenvironment that favored IFN-gamma production tightly correlates with the skin disease in NC mice through the infiltration of eosinophils.     

Characterization of the nuclear import and export functions of Ikappa B(epsilon)

Control over the nuclear localization of nuclear factor kappaB/Rel proteins is accomplished in large part through association with members of the inhibitor of kappaB (IkappaB) protein family. For example, the well studied IkappaBalpha protein actively shuttles between the nucleus and the cytoplasm and both inhibits nuclear import and mediates nuclear export of NF-kappaB/Rel proteins. In contrast, the IkappaBbeta protein can inhibit nuclear import of NF-kappaB/Rel proteins but does not remove NF-kappaB/Rel proteins from the nucleus. To further understand how the IkappaB proteins control the nuclear-cytoplasmic distribution of NF-kappaB/Rel proteins, we have characterized the nuclear import and nuclear export functions of IkappaBepsilon. Our results indicate that the IkappaBepsilon protein, like the IkappaBalpha protein, actively shuttles between the nucleus and the cytoplasm. Similar to IkappaBalpha, nuclear import of IkappaBepsilon is mediated by its ankyrin repeat domain and is not blocked by the dominant-negative RanQ69L protein. However, the nuclear import function of the IkappaBepsilon ankyrin repeat domain is markedly less efficient than that of IkappaBalpha, with the result that nuclear shuttling of IkappaBepsilon between the nucleus and the cytoplasm is significantly slower than IkappaBalpha. Nuclear export of IkappaBepsilon is mediated by a short leucine-rich nuclear export sequence (NES)-like sequence ((343)VLLPFDDLKI(352)), located between amino acids 343 and 352. This NES-like sequence is required for RanGTP-dependent binding of IkappaBepsilon to CRM1. Nuclear accumulation of IkappaB(epsilon) is increased by either leptomycin B treatment or alanine substitutions within the IkappaBepsilon-derived NES. A functional NES is required for both efficient cytoplasmic retention and post-induction control of c-Rel by IkappaBepsilon, consistent with the notion that IkappaBepsilon-mediated nuclear export contributes to control over the nucleocytoplasmic distribution of NF-kappaB/Rel proteins.     

Pyridone 6, a pan-JAK inhibitor, ameliorates allergic skin inflammation of NC/Nga mice via suppression of Th2 and enhancement of Th17

Atopic dermatitis (AD) is a common pruritic inflammatory disease triggered by a defective skin barrier and immunodysregulation. AD has been considered a typical example of a Th2 response associated with allergic disease. In the early phases of the disease, symptoms include IgE hyperproduction, eosinophil accumulation, and mast cell activation; in the chronic phase, a Th1-dominant immune response is also observed at the sites of AD skin lesions. The role of IL-17-producing Th (Th17) cells in AD has not been established. In the current study, we found that pyridone 6 (P6), a pan-JAK inhibitor, delayed the onset and reduced the magnitude of skin disease in an AD-like skin-disease model of NC/Nga mice. P6 reduced IFN-γ and IL-13, whereas it enhanced IL-17 and IL-22 expression. In vitro, P6 also inhibited both Th1 and Th2 development, whereas it promoted Th17 differentiation from naive T cells when present within a certain range of concentrations. This was probably because P6 strongly inhibited STAT1, STAT5, and STAT6 phosphorylation, whereas STAT3 phosphorylation was less efficiently suppressed by P6 at the same concentration. Furthermore, IL-22 protects keratinocytes from apoptosis induced by IFN-γ, and administration of IL-17 and IL-22 partially ameliorated skin diseases in NC/Nga mice. These results suggested that the JAK inhibitor P6 is therapeutic for AD by modulating the balance of Th2 and Th17.     

Low expression of the IL-23/Th17 pathway in atopic dermatitis compared to psoriasis

The classical Th1/Th2 paradigm previously defining atopic dermatitis (AD) and psoriasis has recently been challenged with the discovery of Th17 T cells that synthesize IL-17 and IL-22. Although it is becoming evident that many Th1 diseases including psoriasis have a strong IL-17 signal, the importance of Th17 T cells in AD is still unclear. We examined and compared skin biopsies from AD and psoriasis patients by gene microarray, RT-PCR, immunohistochemistry, and immunofluorescence. We found a reduced genomic expression of IL-23, IL-17, and IFN-gamma in AD compared with psoriasis. To define the effects of IL-17 and IL-22 on keratinocytes, we performed gene array studies with cytokine-treated keratinocytes. We found lipocalin 2 and numerous other innate defense genes to be selectively induced in keratinocytes by IL-17. IFN-gamma had no effect on antimicrobial gene-expression in keratinocytes. In AD skin lesions, protein and mRNA expression of lipocalin 2 and other innate defense genes (hBD2, elafin, LL37) were reduced compared with psoriasis. Although AD has been framed by the Th1/Th2 paradigm as a Th2 polar disease, we present evidence that the IL-23/Th17 axis is largely absent, perhaps accounting for recurrent skin infections in this disease.     

Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions

Skin lesions in the allergic form of atopic dermatitis (AD) are induced by allergen-specific T cells that infiltrate the skin at the site of allergen exposure. Although Th2-type CD4+ T cells appear to be crucial in AD pathophysiology, little is known about the contribution of CD8+ T cells in the development of the allergic skin inflammation. In the present study, we have analyzed the respective role of CD8+ and CD4+ T cells in the development of AD skin lesions in a mouse model of allergen-induced AD. In sensitized mice, CD8+ T cells are rapidly and transiently recruited to the allergen-exposed site and initiate the inflammatory process leading to skin infiltration with eosinophils and Th1/Th2-producing cells. CD8+ T cell-depleted mice show no inflammation, demonstrating that these cells are mandatory for the development of AD. In contrast, CD4+ T cell-depleted mice develop a severe form of eczema. Furthermore, adoptive transfer of CD8+ T cells from sensitized mice into naive recipient mice leads to skin inflammation soon after allergen exposure. These data indicate that allergen-primed CD8+ T cells are required for the development of AD-like lesions in mice.     

TNIP1 reduction sensitizes keratinocytes to post-receptor signalling following exposure to TLR agonists

Cell level inflammatory signalling is a combination of initiation at cell membrane receptors and modulation by cytoplasmic regulatory proteins. For keratinocytes, the predominant cell type in the epidermis, this would include toll-like receptors (TLR) and cytoplasmic proteins that propagate or dampen post-receptor signalling. We previously reported that increased levels of tumor necrosis factor α induced protein 3-interacting protein 1 (TNIP1) in HaCaT keratinocytes leads to decreased expression of stress response and inflammation-associated genes. This finding suggested decreased TNIP1 levels, as seen in some cutaneous disease states, may produce the opposite effect, sensitizing cells to triggers of inflammatory signalling including those sensed by TLR. In this study of TNIP1-deficient HaCaT keratinocytes we examined intracellular signalling consequences especially those expected to produce gene expression changes downstream of TLR3 or TLR2/6 activation by Poly (I:C) or FSL-1, agonists modeling skin relevant pathogens. We found TNIP1-deficient keratinocytes are hyper-sensitive to TLR activation compared to control cells with a normal complement of TNIP1 and receiving the same agonist stimulation. TNIP1-deficient keratinocytes have increased levels of activated (phosphorylated) cytoplasmic mediators such as JNK and p38 and greater nuclear translocation of NF-κB and phospho-p38 when exposed to TLR ligands. This is consistent with significantly increased expression of several inflammatory cytokines and chemokines, such as IL-6 and IL-8. These results describe how decreased TNIP1 levels promote a hyper-sensitive state in HaCaT keratinocytes evidenced by increased activation of signalling molecules downstream of TLR agonists and increased expression of pro-inflammatory mediators. TNIP1 keratinocyte deficiency as reported for some skin diseases may predispose these cells to excessive inflammatory signalling upon exposure to viral or bacterial ligands for TLR.     

Genuine traditional Korean medicine,Naju Jjok (Chung-Dae,Polygonum tinctorium) improves 2,4-dinitrofluorobenzene-induced atopic dermatitis-like lesional skin

PurposeNaju Jjok (NJJ, Polygonum tinctorium) is a clear heat and release toxin medicinal. It has been used to treat various inflammatory diseases and as a dye in clothing in traditional Korean medicine. However, the effect of NJJ on atopic dermatitis (AD) has not been elucidated. Therefore, we examined whether NJJ would have an inhibitory effect on AD using the mimic AD murine model and in vitro model.MethodsWe treated NJJ on 2,4-dinitrofluorobenzene (DNFB)-induced AD-like skin lesions in NC/Nga mice, phorbol myristate acetate/calcium ionophore A23187-stimulated human mast cell line (HMC-1) cells, and anti-CD3/anti-CD28-stimulated splenocytes. Histological analysis, ELISA, PCR, and Western blot analysis were performed.ResultsThe oral administration with NJJ suppressed the total clinical severity in DNFB-induced AD-like lesional skin. NJJ significantly suppressed the levels of inflammatory mRNA and protein in AD-like lesional skin. NJJ significantly suppressed the levels of IgE and interleukin-4 in the serum of DNFB-induced AD mice. The expression of mast cells-derived caspase-1 was suppressed by NJJ in AD-like lesional skin. In addition, topical application with NJJ improved clinical symptoms in DNFB-induced AD mice. The topical application with NJJ significantly suppressed the levels of IgE and histamine in the serum of DNFB-induced AD mice. NJJ suppressed the production and mRNA expression of TSLP by blockade of caspase-1 signal pathway in the activated HMC-1 cells. Furthermore, NJJ significantly decreased the production of tumor necrosis factor-α from the stimulated splenocytes.ConclusionsIn conclusion, these results propose curative potential of natural dye, NJJ by showing the scientific evidence on anti-AD effect of NJJ which has been used traditionally.     

Effect of dendritic cells treated with CpG ODN on atopic dermatitis of Nc/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease and the pathogenesis of AD is associated with the release of various cytokines/chemokines due to activated Th(2) immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide in the context of particular base sequence (CpG motifs) are known to have the immunostimulatory activities in mice and to convert from Th(2) to Th(1) immune responses in AD. We aimed to investigate that CpG ODN, especially phosphodiester form, can stimulate the protective immunity in NC/Nga mice with AD. We isolated BMDCs from NC/Nga mice and then, cultured with GM-CSF and IL-4 for 6 days, and treated for 2 days by either phosphorothioate ODN or phosphodiester ODN. CpG ODN-treated DCs resulted in more production of IL-12. When CpG ODN-treated DCs were intravenously injected into the NC/Nga mice, the NC/Nga mice with CpG ODN-treated DCs showed significant improvement of AD symptoms and decrease of IgE level. Histopathologically, the NC/Nga mice skin with CpG ODN-treated DCs showed the decreased IL-4 and TARC expression comparing with non-injected mice. These results may suggest that phosphodiester CpG ODN-treated DCs might function as a potent adjuvant for AD in a mouse model.