Protective role of copper, zinc superoxide dismutase against UVB-induced injury of the human keratinocyte cell line HaCaT

On the basis of our recent observation that copper, zinc-superoxide dismutase and manganese-superoxide dismutase change differently following a single exposure to ultraviolet-B irradiation in the human keratinocyte cell line HaCaT, we have examined the possible role of endogenous copper,zinc-superoxide dismutase or manganese-superoxide dismutase against ultraviolet-B-induced reactive-oxygen- species-mediated keratinocyte injury in vitro. To evaluate the individual defensive roles of copper, zinc-superoxide dismutase and manganese-super-oxide dismutase, we treated HaCaT cells with diethyldithiocarbamate, a chelating agent of ionic copper that inactivates copper,zinc-superoxide dismutase activities, tumor necrosis factor alpha, which enhances manganese-superoxide dismutase levels, or transforming growth factor beta1, which inhibits manganese-superoxide dismutase levels. After the treatment with each reagent, HaCaT cells in the three different conditions were exposed to a single dose of ultraviolet-B irradiation. We assessed ultraviolet-B-induced cytotoxicity by measuring both lactate dehydrogenase leakage and cell viability using trypan blue dye exclusion assay. The lactate dehydrogenase leakage in the supernatant from damaged HaCaT cells whose copper,zinc-superoxide dismutase levels were inactivated by diethyldithiocarbamate was significantly increased and the cell viability was significantly decreased in comparison with untreated groups at 8 and 24 h after ultraviolet-B irradiation. On the other hand, the lactate dehydrogenase release and cell viability for HaCaT cells whose manganese-superoxide dismutase levels were enhanced by tumor necrosis factor alpha or inhibited by transforming growth factor beta1 showed no significant difference from untreated groups. Furthermore, increased production of intracellular peroxides in HaCaT cells treated with diethyldithiocarbamate was observed by flow cytometric analysis at 8 h after ultraviolet-B irradiation. These results suggest that copper,zinc-superoxide dismutase may play a primary protective role against ultraviolet-B-induced injury of the human keratinocyte cell line HaCaT.     

Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage

Chronic ultraviolet-B irradiation of the skin results in epidermal hyperplasia, degradation of extracellular matrix molecules, and formation of wrinkles. To characterize the biologic role of the vascular system in the mediation of ultraviolet-B-induced skin damage, we performed quantitative analyses of cutaneous blood vessels of mice after 10 wk of ultraviolet-B irradiation. Skin vascularization was greatly increased after chronic ultraviolet-B exposure with a significant increase of both the number and the size of dermal blood vessels, associated with upregulation of vascular endothelial growth factor expression in the hyperplastic epidermis. To directly study whether inhibition of angiogenesis may diminish ultraviolet-B-induced cutaneous damage, wild-type and transgenic mice with skin-specific overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 were subjected to the same ultraviolet-B irradiation regimen. Ultraviolet-B-irradiated thrombospondin-1 transgenic mice showed a significantly reduced skin vascularization, decreased endothelial cell proliferation, and increased endothelial cell apoptosis rates, compared with wild-type mice. Moreover, dermal photo-damage and wrinkle formation were greatly reduced in thrombospondin-1 transgenic mice. These results reveal an important role of the cutaneous vascular system in mediating ultraviolet-B-induced skin damage and suggest inhibition of angiogenesis as a potential new approach for the prevention of chronic cutaneous photo-damage.     

Dermal fibroblasts sustain proliferation of activated T cells via membrane-bound interleukin-15 upon long-term stimulation with tumor necrosis factor-alpha

In chronic inflammatory conditions, mononuclear cells infiltrate the connective tissue attracted by fibroblast-secreted chemokines. The role of fibroblasts in sustaining the lymphocyte immune response upon cellular infiltration is so far unresolved. We here report that, upon prolonged stimulation with tumor necrosis factor-alpha, dermal fibroblasts enhance proliferation of activated T cells whereas unstimulated fibroblasts do not. T cell growth stimulation requires cell contact of tumor necrosis factor-alpha stimulated fibroblasts to T cells and is not due to soluble factors. Growth stimulation is substantially blocked by neutralizing antibodies to interleukin-15. Fluorescence-activated cell sorter analyses revealed that tumor necrosis factor alpha stimulated fibroblasts expose interleukin-15 in a membrane-bound form on the cell surface whereas nonstimulated fibroblasts and interferon-gamma treated fibroblasts do not. The amount of membrane interleukin-15 increases with the duration of tumor necrosis factor-alpha stimulation for at least 3 d. Unstimulated fibroblasts, however, accumulate interleukin-15 in the cytoplasm. No interleukin-15 could be detected in the culture supernatant. Immunohistochemical analyses confirmed membrane interleukin-15 on dermal fibroblasts in discoid lupus erythematosus skin lesions whereas no membrane interleukin-15 was found on the surface of fibroblasts in healthy skin. We conclude that dermal fibroblasts upon long-term tumor necrosis factor-alpha stimulation during chronic inflammation are involved via membrane-bound interleukin-15 in stimulating proliferation of accumulated, activated T cells.     

Inflammatory skin disease in K14/p40 transgenic mice: evidence for interleukin-12-like activities of p40

The proinflammatory cytokine interleukin-12, a p35/p40 heterodimer, is produced by resident cells in skin and has been implicated as a pathogenetic factor in T-cell-mediated skin diseases. Secretion of heterodimeric interleukin-12 is always accompanied by production of p40 monomer and p40/p40 homodimer. To investigate the possible in vivo role of p40 per se, we generated mice that constitutively express monomeric and homodimeric p40 in basal keratinocytes. These mice spontaneously developed an eczematous skin disease that was characterized by hyperkeratosis, focal epidermal spongiosis, and a mixed inflammatory infiltrate composed of T cells (CD4+), macrophages, eosinophils, mast cells, and few neutrophils. Fluorescence-activated cell sorter analysis of transgenic epidermal cell suspensions revealed induction of major histocompatibility complex class II molecules on keratinocytes and a 2-3-fold increase in the content of Langerhans cells. Cytokines produced by these activated epidermal cells include interleukin-1alpha and tumor necrosis factor alpha. The skin disease in K14/p40 mice was similar to that of littermate mice that received injections of interleukin-12, suggesting overlapping in vivo functional properties. As induction of interferon-gamma is a major function of interleukin-12, we tested the in vitro ability of transgenic p40 to induce interferon-gamma. In contrast to interleukin-12, transgenic p40 did not stimulate interferon-gamma secretion by cultured splenocytes. We conclude that transgenic p40 and interleukin-12 are equally capable of initiating cutaneous inflammation. Despite these in vivo similarities, there is a clear functional difference between interleukin-12 and transgenic p40 in vitro, suggesting that interferon-gamma is not a major factor contributing to interleukin-12-like activities of transgenic p40.     

Differential expression of cytokine mRNA in skin specimens from patients with erythema migrans or acrodermatitis chronica atrophicans

Erythema migrans, the characteristic skin manifestation of acute Lyme borreliosis, is a self-limited lesion. In contrast, acrodermatitis chronica atrophicans, the typical cutaneous manifestation of late Lyme borreliosis, is a chronic skin condition. In an effort to understand pathogenic factors that lead to different outcomes in dermatoborrelioses, skin biopsy samples from 42 patients with erythema migrans and 27 patients with acrodermatitis chronica atrophicans were analyzed for mRNA expression of five pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interferon-gamma, and interleukin-2) and two anti-inflammatory cytokines (interleukin-4 and interleukin-10) by in situ hybridization with cytokine-specific riboprobes. Among the 27 patients who had erythema migrans alone with no associated signs or symptoms, the major cytokines expressed in perivascular infiltrates of T cells and macrophages were the pro-inflammatory cytokine interferon-gamma and the anti-inflammatory cytokine interleukin-10. In the 15 erythema migrans patients who had associated signs and symptoms, including headache, elevated temperature, arthralgias, myalgias, or fatigue, a larger number of macrophages and greater expression of macrophage-derived pro-inflammatory cytokines, tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6, were also found. In comparison, infiltrates of T cells and macrophages in the skin lesions of acrodermatitis chronica atrophicans patients had very little or no interferon-gamma expression. Instead, they usually expressed only the pro-inflammatory cytokine tumor necrosis factor alpha and the anti-inflammatory cytokine interleukin-4. Thus, the activation of pro-inflammatory cytokines in erythema migrans lesions, particularly interferon-gamma, seems to be important in the control of the spirochetal infection. In contrast, the restricted pattern of cytokine expression in acrodermatitis chronica atrophicans, including the lack of interferon-gamma, may be less effective in spirochetal killing, resulting in the chronicity of this skin lesion. J Invest Dermatol 115:1115-1123 2000     

Suppressed alloantigen presentation, increased TNF-alpha, IL-1, IL-1Ra, IL-10, and modulation of TNF-R in UV-irradiated human skin

Cytokines induced in skin by ultraviolet radiation cause local and systemic immunosuppression. Tumor necrosis factor alpha, interleukin-1, and interleukin-10 are key mediators in the mouse, but less is known about cytokine synthesis and function in ultraviolet-irradiated human skin. We exposed human skin to 3 minimal erythema doses of solar-simulated radiation and raised suction blisters at intervals to 72 h. Alloantigen presentation was suppressed in a mixed epidermal cell-lymphocyte reaction by 69% from 4 to 15 h post-solar-simulated radiation, but recovered to control values by 24 h. Tumor necrosis factor alpha was raised at 4 h after solar-simulated radiation, reached a maximum 8-fold increase at 15 h, then rapidly declined to control values. Interleukin-1alpha and interleukin-1beta were first increased at 15 h, and remained raised to 72 h, although interleukin-1beta declined from its 15 h maximum. Interleukin-10 increased a maximum 2-fold between 15 and 24 h, coincident with recovery of mixed epidermal cell-lymphocyte reaction responses and downregulation of tumor necrosis factor alpha and interleukin-1beta. Solar-simulated radiation differentially affected soluble tumor necrosis factor alpha receptors; soluble tumor necrosis factor-RI was suppressed 33% at 8-15 h whereas soluble tumor necrosis factor-RII increased 2-fold from 15 to 48 h. Interleukin-1 receptor antagonist was raised at all times post-irradiation. Interleukin-12 was not detectable in control or irradiated skin. These kinetics suggest the tumor necrosis factor alpha network has primary importance in ultraviolet-damaged human skin. The small increase in interleukin-10 implies that 3 minimal erythema doses of solar-simulated radiation is the threshold dose for its induction and local, rather than systemic, functions for interleukin-10 in immunosuppression and regulation of other cytokines.     

Inhibition of erbB receptor family members protects HaCaT keratinocytes from ultraviolet-B-induced apoptosis

In the human epidermis, the cells most at risk for the development of cancer due to sunlight exposure are the keratinocytes. In animal models, ultraviolet-B is a complete carcinogen, capable of inducing and promoting the development of malignant cells. A key element of ultraviolet-B-induced carcinogenesis is the ability of ultraviolet-B to induce the expression of a number of cellular proteins and activate growth factor receptor tyrosine kinases, including the erbB receptor family. Keratinocytes express the erbB1 (also called EGF-R, HER1), the erbB2 (also known as neu or HER2), and the erbB3 (HER3) subtypes. In general, activation of the erbB receptor family leads to a cellular proliferative response. In certain instances, however, activation of an erbB receptor can induce differentiation, cell cycle arrest, and even apoptosis. The inhibition of tyrosine kinase activity in rodent models and human skin has been shown to inhibit some ultraviolet-B response pathways. We have shown that the inhibition of erbB receptors, by both pharmaceutical and immunologic means, will inhibit ultraviolet-B-induced apoptosis in the HaCaT human keratinocyte cell line. This inhibition was specific for the erbB receptor family and specific for ultraviolet-B-induced apoptosis. These results suggest that, in certain instances, ultraviolet-B-induced apoptotic signaling requires erbB family receptor activity.     

The skin: an immunoreactive target organ during interleukin-2 administration?

Six patients with metastatic malignant melanoma were treated systemically with dacarbazine and interleukin-2 (IL-2). During IL-2 administration all patients developed a macular erythematous rash followed by scaling which began 24-48 h after IL-2 infusion. The dermatological changes were associated with elevated interferon-gamma and tumor necrosis factor alpha serum levels (immunoradiometric assay). Histology revealed nonspecific spongiotic foci in the epidermis and a perivascular mononuclear infiltrate in the dermis. Immunohistochemistry characterized this infiltrate mainly as activated T helper lymphocytes and revealed the expression of intercellular adhesion molecule 1 by endothelial cells and keratinocytes that might have been induced by interferon-gamma. The skin reactions associated with systemic IL-2 administration, show that the skin actually participates as a target organ. They should be differentiated from drug eruptions.     

Associations of tumor necrosis factor alpha and HLA polymorphisms with adult dermatomyositis: implications for a unique pathogenesis

We recently reported that the -308A tumor necrosis factor alpha promoter polymorphism is associated with the photosensitive disorder subacute cutaneous lupus erythematosus and mediates an exaggerated tumor necrosis factor alpha response to ultraviolet B. We now sought to examine the association of this polymorphism with adult dermatomyositis, a photosensitive disease that exhibits some features in common with subacute cutaneous lupus erythematosus. Fifty adult patients with dermatomyositis and 239 healthy, race-matched controls were examined for the -308A tumor necrosis factor alpha polymorphism and the more common -308G allele. The frequency of the -308A allele was 0.27 in the entire dermatomyositis group, versus 0.14 in the controls (p = 0.003, chi2 2 x 2 table). Caucasians were the only racial/ethnic group in our study large enough to allow separate statistical analysis (47 dermatomyositis, 223 controls). The frequency of the -308A allele was 0.26 for dermatomyositis and 0.14 for controls (p = 0.014). Caucasians are known to exhibit a linkage disequilibrium between -308A and HLA-DR3, which we previously found to be significantly enhanced in subacute cutaneous lupus erythematosus patients. In contrast, we now found no increase in the association of -308A and HLA-DR3 in Caucasians with dermatomyositis compared to controls. Consistent with this observation, the association of these two genes in dermatomyositis was significantly less than we previously reported in Caucasians with subacute cutaneous lupus erythematosus (p = 0.016). We conclude that the tumor necrosis factor -308A polymorphism is associated with dermatomyositis, which suggests a pathophysiologic contribution from ultraviolet-induced production of tumor necrosis factor alpha, similar to subacute cutaneous lupus erythematosus. The differences in linkage with HLA-DR3, as well as several divergent clinical features, indicate that there are also fundamental mechanistic differences between dermatomyositis and subacute cutaneous lupus erythematosus.     

Wavelength-specific synergy between ultraviolet radiation and interleukin-1 alpha in the regulation of matrix-related genes: mechanistic role for tumor necrosis factor-alpha.

Ultraviolet light causes both acute and chronic changes in extracellular matrix. We sought to examine the effects of different ultraviolet wavelengths on expression of matrix-related genes in fibroblasts. We previously reported that tropoelastin gene expression in vivo decreases with acute ultraviolet B exposure, and interleukin-1 alpha-mediated upregulation of tropoelastin is blocked in vitro after ultraviolet B radiation. In this study, we found that only ultraviolet B, but not ultraviolet A or ultraviolet A1, blocked the ability of interleukin-1 alpha to stimulate tropoelastin expression in vitro. Ultraviolet B and interleukin-1 alpha synergistically increased tumor necrosis factor-alpha secretion by fibroblasts, a finding not seen with ultraviolet B alone nor with ultraviolet A or ultraviolet A1 combined with interleukin-1 alpha. Keratinocytes showed a similar ultraviolet B-specific induction of tumor necrosis factor-alpha production. Addition of tumor necrosis factor-alpha to cultured fibroblasts blocked interleukin-1 alpha-induced stimulation of tropoelastin message, and addition of anti-tumor necrosis factor-alpha antibodies restored the responsiveness of tropoelastin and collagen messages to exogenous interleukin-1 alpha after ultraviolet B exposure. We conclude that interleukin-1 alpha in combination specifically with ultraviolet B induces fibroblasts to secrete tumor necrosis factor-alpha, and that this ultraviolet B-specific induction of tumor necrosis factor-alpha secretion is responsible for effects of ultraviolet B on the expression of matrix-related genes in the skin.     

A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin

Atopic dermatitis is an inflammatory skin disease in which the inflammation is characterized by the influx of lymphocytes into the dermis. It is generally believed that atopic dermatitis is a Th2-type disease, i.e., the T lymphocytes produce interleukin-4, interleukin-5, interleukin-10, and interleukin-13, although it has become evident in recent years that the cytokine profile in the skin changes during the course of the disease towards a Th1-Th2 mixed cytokine profile (interferon-gamma, tumor necrosis factor alpha, and interleukin-2). The lymphocytes that home into the skin express cutaneous lymphocyte-associated antigen, and it has recently been shown that most of the lymphocytes in this population express the chemokine receptor CCR4. CCR4 is the receptor for the CC chemokine TARC (thymus and activation regulated chemokine), and this chemokine is expressed predominantly by keratinocytes in the basal layer of the epidermis of lesional atopic dermatitis skin in mice. In humans, however, it was shown to be expressed in the endothelial cells of the dermis. We have examined the peripheral blood mononuclear cells of atopic dermatitis patients for the expression of cutaneous lymphocyte-associated antigen and CCR4 and compared them with peripheral blood mononuclear cells from normal controls. We found that the proportion of CLA+CCR4+ lymphocytes is upregulated in atopic dermatitis patients. In addition we have examined skin biopsies of lesional and non-lesional skin from atopic dermatitis patients and found that the keratinocytes, but not the endothelial cells, produce TARC in the lesional but not in the nonlesional skin. To gain insight in the stimulatory mechanisms for TARC production in keratinocytes, as previously observed in mice, we cultured HaCaT cells and found that interferon-gamma and tumor necrosis factor alpha work synergistically to induce TARC production. These observations suggest that the induction of TARC production in keratinocytes plays an important role in the late phase skin invasion by CCR4+CLA+ Th2-type lymphocytes in atopic dermatitis.     

Evidence for involvement of the epidermal platelet-activating factor receptor in ultraviolet-B-radiation-induced interleukin-8 production

Ultraviolet B radiation has been shown to generate cutaneous inflammation in part through inducing oxidative stress and cytokine production in human keratinocytes. Amongst the proinflammatory cytokines synthesized in response to ultraviolet B radiation is the potent chemoattractant interleukin-8. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in ultraviolet-B-induced epidermal cell cytokine production. These studies examined the role of the PAF system in ultraviolet-B-induced epidermal cell interleukin-8 biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF-receptor-negative human epidermal cell line KB with the human PAF receptor. Treatment of PAF-receptor-expressing KB cells with the metabolically stable PAF receptor agonist carbamoyl-PAF resulted in increased interleukin-8 mRNA and protein, indicating that activation of the epidermal PAF receptor was linked to interleukin-8 production. Ultraviolet B irradiation of PAF-receptor-expressing KB cells resulted in significant increases in both interleukin-8 mRNA and protein in comparison to ultraviolet-B-treated control KB cells. Pretreatment with PAF receptor antagonists inhibited both carbamoyl-PAF-induced and ultraviolet-B-induced interleukin-8 production in the PAF-receptor-positive cells, but not in control KB cells. Similarly, treatment of the PAF-receptor-expressing primary cultures of human keratinocytes or the human epidermal cell line A-431 with carbamoyl-PAF or ultraviolet B radiation resulted in interleukin-8 production that was partially inhibited by PAF receptor antagonists. These studies suggest that the epidermal PAF receptor may be a pharmacologic target for ultraviolet B radiation in skin and thus may act to augment ultraviolet-B-mediated production of cytokines such as interleukin-8.