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.     

Ultraviolet B radiation-induced cell death: critical role of ultraviolet dose in inflammation and lupus autoantigen redistribution

The nuclear self-Ags targeted in systemic lupus erythematosus translocate to the cell membrane of UV-irradiated apoptotic keratinocytes and may represent an important source of self-immunization. It is hard to understand how the noninflammatory milieu accompanying most apoptosis might provoke an immunogenic response leading to autoantibodies. We have found that the precise amount of keratinocyte UV exposure is crucial in determining the rate of apoptosis, the amount of inflammatory cytokine production, and the degree of autoantigen translocation. Low doses of UVB (相似文献   

Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin

The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm2 UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1alpha levels increased with LOM concentration. The release of TNF-alpha and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 micromol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM.     

In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-gamma-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-gamma production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-gamma-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-gamma, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.     

Eccrine Sweat Contains IL-1α, IL-1β and IL-31 and Activates Epidermal Keratinocytes as a Danger Signal

Eccrine sweat is secreted onto the skin''s surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin''s surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.     

Crosstalk between keratinocytes and T lymphocytes via Fas/Fas ligand interaction: modulation by cytokines.

Apoptosis mediated by Fas/FasL interaction plays an important role during many inflammatory skin disorders. To estimate whether the expression of FasL, the ligand for Fas, might be regulated by cytokines we stimulated primary human keratinocytes with several pro- and anti-inflammatory cytokines. Keratinocytes cultured to subconfluence expressed FasL constitutively. Cells stimulated with the proinflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-15, respectively, increased significantly their intracellular as well as cell surface-bound FasL expression in a time- and dose-dependent manner. This cytokine-induced FasL expression was dependent on new protein synthesis. Despite enhanced expression of cell surface-bound FasL, no release of soluble FasL was measured in the cell supernatants determined by ELISA. Stimulation of the cells with IL-6, IL-10, IL-12, TGF-beta1, and GM-CSF did not modulate the constitutive FasL expression, but IFN-gamma-mediated FasL up-regulation was significantly diminished by IL-10 and TGF-beta1, respectively. Up-regulation of FasL on IFN-gamma-stimulated keratinocytes led to increased apoptosis within monolayers cultured for 48 h. Moreover, coculture experiments performed with Fas+ Jurkat T cells revealed that enhanced FasL expression on IFN-gamma-stimulated keratinocytes induced apoptosis in cocultured T cells, demonstrating that up-regulated FasL was functionally active. In summary, our data suggest the important regulatory role of cytokine-controlled Fas/FasL interaction in the cross-talk between keratinocytes and skin-infiltrating T cells for maintenance of homeostasis in inflammatory skin processes.     

IL-4 suppresses UVB-induced apoptosis in skin

In this study, cutaneous role of IL-4 in UVB-induced apoptosis was investigated using transgenic mice with skin-specific expression of IL-4 (IL-4 Tg mice). The transgenic mice did not show any gross clinical abnormalities. However, epidermis was thickened and increased MHC class II positive cells were detected as well as enhanced expression of inflammatory cytokines such as IL-1 and TNF-alpha in skin. In addition, histological analysis revealed increased infiltration of lymphocytes, acanthosis, hyperkeratosis, and parakeratosis in skin of IL-4 Tg mice. The physiological effect of IL-4 overexpression in skin against environmental stimulus such as UVB was investigated by irradiating wild-type and IL-4 Tg mice with UVB followed by evaluation of apoptosis. The result demonstrated suppressed apoptosis in epidermis of IL-4 Tg mice compared with wild-type mice. To further assess anti-apoptotic function of IL-4 in keratinocytes, stable cell clones were made where IL-4 was constitutively overexpressed and examined for UVB-induced apoptosis. The results showed that apoptosis was remarkably decreased in IL-4 over-expressing cell clones compared with that in mock transfected cells. Collectively, data presented here shows that IL-4 has an inhibitory effect against UVB-induced apoptosis in keratinocytes, suggesting that IL-4 may be an important regulator in cutaneous immunity against UVB.     

Roles of TLR7 in Activation of NF-κB Signaling of Keratinocytes by Imiquimod

Imiquimod is known to exert its effects through Toll-like receptor 7 (TLR7) and/or TLR8, resulting in expression of proinflammatory cytokines and chemokines. Keratinocytes have not been reported to constitutively express TLR7 and TLR8, and the action of imiquimod is thought to be mediated by the adenine receptor, not TLR7 or TLR8. In this study, we revealed the expression of TLR7 in keratinocytes after calcium-induced differentiation. After addition of calcium to cultured keratinocytes, the immunological responses induced by imiquimod, such as activation of NF-κB and induction of TNF-α and IL-8, were more rapid and stronger. In addition, imiquimod induced the expression TLR7, and acted synergistically with calcium to induce proinflammatory cytokines. We confirmed that the responses induced by imiquimod were significantly inhibited by microRNAs suppressing TLR7 expression. These results suggest that TLR7 expressed in keratinocytes play key roles in the activation of NF-κB signaling by imiquimod, and that their modulation in keratinocytes could provide therapeutic potential for many inflammatory skin diseases.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

FcR-independent effects of IgE and IgG autoantibodies in bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by IgE and IgG class autoantibodies specific for 180-kDa BP Ag 2 (BP180), a protein involved in cell-substrate attachment. Although some direct effects of BP IgG have been observed on keratinocytes, no study to date has examined direct effects of BP IgE. In this study, we use primary cultures of human keratinocytes to demonstrate Ag-specific binding and internalization of BP IgE. Moreover, when BP IgE and BP IgG were compared, both isotypes stimulated FcR- independent production of IL-6 and IL-8, cytokines critical for BP pathology, and elicited changes in culture confluence and viability. We then used a human skin organ culture model to test the direct effects of these Abs on the skin, whereas excluding the immune inflammatory processes that are triggered by these Abs. In these experiments, physiologic concentrations of BP IgE and BP IgG exerted similar effects on human skin by stimulating IL-6 and IL-8 production and decreasing the number of hemidesmosomes localized at the basement membrane zone. We propose that the Ab-mediated loss of hemidesmosomes could weaken attachment of basal keratinocytes to the basement membrane zone of affected skin, thereby contributing to blister formation. In this article, we identify a novel role for IgE class autoantibodies in BP mediated through an interaction with BP180 on the keratinocyte surface. In addition, we provide evidence for an FcR-independent mechanism for both IgE and IgG class autoantibodies that could contribute to BP pathogenesis.     

Sustained release of Smac/DIABLO from mitochondria commits to undergo UVB-induced apoptosis

Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m², but not at 150 J/m², induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m² UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m² UVB, but not at 150 J/m². However, with both 150 and 500 J/m² UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m² UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.     

Human primary keratinocytes show restricted ability to up-regulate suppressor of cytokine signaling (SOCS)3 protein compared with autologous macrophages

Suppressor of cytokine signaling (SOCS)3 belongs to a family of proteins that are known to exert important functions as inducible feedback inhibitors and are crucial for the balance of immune responses. There is evidence for a deregulated immune response in chronic inflammatory skin diseases. Thus, it was the aim of this study to investigate the regulation of SOCS proteins involved in intracellular signaling pathways occurring during inflammatory skin diseases and analyze their impact on the course of inflammatory responses. Because we and others have previously described that the cytokine IL-27 has an important impact on the chronic manifestation of inflammatory skin diseases, we focused here on the signaling induced by IL-27 in human primary keratinocytes compared with autologous blood-derived macrophages. Here, we demonstrate that SOCS3 is critically involved in regulating the cell-specific response to IL-27. SOCS3 was found to be significantly up-regulated by IL-27 in macrophages but not in keratinocytes. Other STAT3-activating cytokines investigated, including IL-6, IL-22, and oncostatin M, also failed to up-regulate SOCS3 in keratinocytes. Lack of SOCS3 up-regulation in skin epithelial cells was accompanied by prolonged STAT1 and STAT3 phosphorylation and enhanced CXCL10 production upon IL-27 stimulation compared with macrophages. Overexpression of SOCS3 in keratinocytes significantly diminished this enhanced CXCL10 production in response to IL-27. We conclude from our data that keratinocytes have a cell type-specific impaired capacity to up-regulate SOCS3 which may crucially determine the course of chronic inflammatory skin diseases.     

Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm²× 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.     

Protective Effect of Indole-3-Pyruvate against Ultraviolet B-Induced Damage to Cultured HaCaT Keratinocytes and the Skin of Hairless Mice

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm²) and maintained with or without test compounds (1–25 mM). In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (100 µmol) and exposed to UVB light (1 J/cm²) for two times. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.     

Upregulation of Cell-Surface-Associated Plasminogen Activation in Cultured Keratinocytes by Interleukin-1βand Tumor Necrosis Factor-α

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA) which is bound in an autocrine manner to a specific receptor (uPA-R) at the keratinocyte surface. Plasminogen that is also bound to specific membrane binding sites is readily activated by uPA-R-bound uPA. Thus, plasmin is provided for proteolysis of pericellular glycoproteins. The expression of uPA and the uPA-R is confined to migrating keratinocytes during epidermal wound healing, rather than to keratinocytes of the normal epidermis. The regulatory factors of uPA/uPA-R expression in keratinocytes remained largely elusive. Proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β), are present in epidermal wounds. We have therefore tested IL-1β and TNF-α for their influence on surface-associated plasminogen activation in a human keratinocyte cell line (HaCaT) as well as in primary cultures of normal human epidermal keratinocytes. Both cytokines induced the secretion of uPA into the culture supernatants and a concomitant increase in uPA activity as well as in uPA and uPA-R antigen at the cell surface. The increase was preceded by an increase in specific mRNA. The induction was accompanied by an accelerated uPA-dependent and plasmin-mediated detachment of HaCaT cells from the culture substratum. Taken together, the proinflammatory cytokines IL-1β and TNF-α induced a coordinated increase in uPA and uPA-R as well as increased pericellular plasmin-mediated proteolysis in human epidermal keratinocytes. This function might be an element of the molecular cell biological events during epidermal wound healing.     

Cytokine Degradation by Biofilms of Porphyromonas gingivalis

The aim of this study was to determine whether biofilms of Porphyromonas gingivalis could proteolytically degrade the cytokines interleukin (IL)-1β, IL-6, or IL-1 receptor antagonist (IL-1ra). Biofilms were grown on membrane filters on the surface of Wilkins-Chalgren blood agar. The biofilms were removed from the plates, and solutions containing 2.5 μg/ml of each cytokine were added. Following incubation for up to 4.0 h, supernatants from the biofilms were subjected to SDS-PAGE. The separated proteins were transferred by Western blotting to PVDF membranes and probed with peroxidase-conjugated antibodies recognizing both the intact cytokines and their degradation products. After 2 h, no intact IL-1β, IL-6, or IL-1ra were detectable. Cytokine proteolysis also occurred in the presence of horse serum. These results demonstrate that biofilm-grown P. gingivalis can degrade both pro- and anti-inflammatory cytokines and so may be able to perturb cytokine networks in vivo by eliminating cytokines from the local environment. Received: 12 August 1997 / Accepted: 15 October 1997