CD11b+ cells are the major source of oxidative stress in UV radiation-irradiated skin: possible role in photoaging and photocarcinogenesis

Exposure of skin to solar UV radiation induces oxidative stress and suppression of cell-mediated immune responses. These effects are associated with the greater risk of several skin disorders including photoaging and photocarcinogenesis. We have shown that UV-induced infiltrating leukocytes contribute in developing oxidative stress in UV-irradiated skin. The peak period of UV-induced infiltrating leukocytes lies between 48 and 72 h after UV exposure of the skin. In this study we demonstrated that UV (90 mJ/cm2)-induced infiltrating CD11b+ cells in C3H/HeN mice skin were the major source of oxidative stress. Hydrogen peroxide (H2O2) was determined as a marker of oxidative stress. Flow cytometric analysis of viable cells revealed that the number of CD11b+H2O2+ cells were significantly higher (31.8%, P < 0.001) in UV-irradiated skin in comparison with non-UV-exposed skin (0.4%). Intraperitoneal administration of monoclonal antibodies to CD11b (rat IgG2b) to C3H/HeN mice inhibited UVB-induced infiltration of leukocytes, as evidenced by reduction in myeloperoxidase activity (64-80%, P < 0.0005), concomitant with significant reduction in H2O2 production both in epidermis and dermis (66-83%, P < 0.001-0.0005) when compared with the administration of rat IgG2b isotype of anti-CD11b. Furthermore, CD11b+ and CD11b- cell subsets were separated by immunomagnetic cell isolation technique from total epidermal and dermal single cell suspensions obtained 48 h after UV irradiation of the skin and analyzed for H2O2 production. Analytical data revealed that CD11b+ cell population from UV-irradiated skin resulted in significantly higher production of total H2O2 in both epidermis and dermis (87-89%, P < 0.0001) in comparison with CD11b- cell population (11-13% of total H2O2). These data revealed that infiltrating CD11b+ cells were the major source of oxidative stress in UV-irradiated skin and thus may contribute to photoaging and promotion of skin tumor growth within the UV-irradiated skin. Together, these data suggest that reduction in UV-induced skin infiltration of CD11b+ cells may be an alternative and effective strategy to reduce solar UV light-induced oxidative stress-mediated skin disorders including photoaging and photocarcinogenesis.     

Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis

It is well known that UV exposure of human skin induces DNA damage, and the cumulative effect of such repeated damage is an important contributor to the development of skin cancer. Here, we demonstrate UV dose- and time-dependent induction of DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in skin cells following a single exposure of human skin to UV radiation. CPD+ cells were identified by an immunohistochemical technique using monoclonal antibodies to thymine dimers. The percentage of CPD+ cells was UV dose-dependent, even a suberythemal (0.5 minimal erythemal dose [MED]) dose resulted in detectable level of cells that contained pyrimidine dimers. Forty-eight hours after irradiation the percent of total epidermal cells positive for CPD ranged from 19 +/- 8, 36 +/- 10, 57 +/- 12 and 80 +/- 10, and total percent dermal cells positive for CPD ranged from 1 +/- 1, 7 +/- 3, 16 +/- 3 and 20 +/- 5, respectively, following 0.5, 1.0, 2.0 and 4.0 MED. CPD were also observed in deeper reticular dermis, which suggest the penetrating ability of UV radiation into the skin. The change in CPD+ cells from 0.5 to 240 h post-UV exposure in both epidermal and dermal compartments of the skin was also quantitated. CPD+ cells were observed in skin biopsies at early time points after UV exposure which remained elevated for 48 h, then declined significantly by 3 days post-UV. A close examination of the skin at and after 3 days following UV exposure indicates the significant removal of DNA damaged cells from the epidermis. Ten days after UV exposure the levels of CPD+ cells in both epidermis and dermis were not significantly different from that in unirradiated skin.     

Molecular responses to stress induced in normal human caucasian melanocytes in culture by exposure to simulated solar UV

Melanocytes play a central role in the response of skin to sunlight exposure. They are directly involved in UV-induced pigmentation as a defense mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The transformation process whereby UV damage may result in melanoma initiation is poorly understood, especially in terms of UV-induced genotoxicity in pigmented cells, where melanin can act either as a sunscreen or as a photosensitizer. The aim of this study was to analyze the behavior of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to simulated solar UV (SSUV, 300-400 nm). Even at relatively high doses (until 20 min exposure, corresponding to 12 kJ/m2 UV-B and 110 kJ/m2 UV-A), cell death was limited, as shown by cell viability and low occurrence of apoptosis (caspase-3 activation). Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2-M phase, and this correlated well with a high induction level of the gene GADD45, 4 h after exposure. Among the genes involved in DNA repair, gene XPC was the most inducible because its expression increased more than two-fold 15 h after a 20 min exposure, whereas expression of P48 was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was also observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UV-A radiation only (320-400 nm), and stimulation of melanogenesis before irradiation further increased HO1 induction. These results were obtained with normal human cells after exposure to SSUV radiation, which mimicked natural sunlight. They provide new data related to gene expression and suggest that melanin in light skin could contribute to sunlight-induced genotoxicity and maybe to melanocyte transformation.     

Differential roles of T-cell subsets in regulation of ultraviolet radiation induced cutaneous photocarcinogenesis

Ultraviolet (UV) radiation, in particular the midwavelength range (UVB; 290-320 nm), is one of the most significant risk factors for the development of nonmelanoma skin cancer. UVB radiation-induced immunosuppression, which occurs in both humans and laboratory animals, contributes to their pathogenesis. However, there are conflicting reports on the relative role of CD4(+) and CD8(+) T cells in UVB induced skin cancer. The purpose of this study was to delineate the contribution of these two cell subpopulations to UVB induced immunosuppression and tumor development using C3H/HeN (WT), CD4 knockout (CD4(-/-) ) and CD8 knockout (CD8(-/-) ) mice. We observed that UVB induced skin carcinogenesis was retarded in terms of number of tumors per group, tumor volume and percentage of mice with tumors, in mice deficient in CD4(+) T cells compared with wild-type mice, whereas significantly greater (P < 0.05) numbers of tumors occurred in CD8(-/-) mice. These results indicate that, CD4(+) T cells promote tumor development while CD8(+) T cells have the opposite effect. Further, we found that CD4(+) T cells from tumor-bearing mice produced interleukin (IL)-4, IL-10, and IL-17 whereas CD8(+) T cells produced interferon-γ. Manipulation of T-cell subpopulations that are induced by UVB radiation could be a means of preventing skin cancers caused by this agent.     

INHIBITION OF 12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED TUMOR PROMOTION IN MURINE SKIN BY SYSTEMIC EFFECTS OF ULTRAVIOLET IRRADIATION

Systemic effects of UVB irradiation (280-320 nm) have been shown to prevent subsequent chemical tumorigenesis induced by an initiation-promotion protocol. The present investigation was designed to determine whether initiation or promotion is prevented by UV irradiation. Groups of 25 B6D2F1/J mice received 12 weeks of intermittent dorsal UVB radiation treatments administered before, or 3 weeks after, initiation with a single application of 7,12-dimethylbenz[a]anthracene on the ventral skin. All mice were promoted ventrally with 5 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) applied three times weekly throughout the experiment. UV irradiation consisted of five 30-min exposures per week to a bank of 6 Westinghouse FS40 sunlamps. UV irradiation applied before or after initiation resulted in a decrease of 18-16 tumors per group of 25 mice, for a reduction of 61 and 50%, respectively, at 24 weeks after the first TPA treatment. Thus, prevention of tumor development was similar whether the UV influence was present or not during initiation. This finding suggests that the UV prevention of promotion could account for UV inhibition of skin tumors induced by an initiation-promotion regimen. Consistent with this concept, pretreatment of mice with dorsal UVB radiation was found to reduce DNA synthesis after exposure to TPA by 46%, although it did not decrease tritiated benzo[a]pyrene binding to DNA, in ventral epidermis. Thus, UVB irradiation systemically reduced TPA-induced tumor promotion in murine skin.     

Silymarin, a flavonoid from milk thistle (Silybum marianum L.), inhibits UV-induced oxidative stress through targeting infiltrating CD11b+ cells in mouse skin

Phytochemicals have shown promise in inhibiting UV-induced oxidative stress, and therefore are considered as potent inhibitors of UV-induced oxidative stress-mediated skin diseases. We have shown previously that topical treatment of silymarin, a flavonoid from milk thistle (Silybum marianum), inhibits UV-induced oxidative stress in mouse skin. However, the cellular targets responsible for the inhibition of UV-induced oxidative stress by silymarin are not clearly defined. To address this issue, C3H/HeN mice were UV irradiated (90 mJ cm(-2)) with or without topical treatment with silymarin (1 mg cm(-2) skin area). Mice were killed 48 h later and skin samples collected. Flow cytometric analysis of viable dermal cells revealed that the number of infiltrating CD11b+ cells were the major source of oxidative stress (31.8%) in UV-irradiated skin compared with non-UV-exposed skin (0.4%). Treatment of silymarin inhibited UV-induced oxidative stress through inhibition of infiltrating CD11b+ cells. The analysis of myeloperoxidase also indicated that silymarin significantly (P < 0.001) decreased UV-induced infiltration of leukocytes, and this effect of silymarin was similar to that of intraperitoneal treatment of mice with monoclonal antibodies to CD11b. The inhibitory effect of silymarin, regardless of whether it is topically treated before or after UV irradiation, was of similar magnitude. Intraperitoneal administration of monoclonal antibodies to CD11b (rat IgG2b) to C3H/HeN mice inhibited UVB-induced oxidative stress generated by both epidermal and dermal cells as is evident by relative fluorescence intensity of oxidized rhodamine. Similar to the effect of anti-CD11b, silymarin also inhibited UV-induced oxidative stress in both epidermal and dermal cells. Further, CD11b+ and CD11b- cell subsets from UV-treated or silymarin+UV-treated mice were separated by immunomagnetic cell isolation technique from total epidermal and dermal single cell suspensions and analyzed for reactive oxygen species (ROS)/H2O2 production. Analytic data revealed that CD11b+ cell population from UV-irradiated skin resulted in significantly higher production of ROS in both epidermis and dermis than CD11b- cell population, and that silymarin inhibited UV-induced oxidative stress through targeting infiltrating the CD11b+ cell type in the skin.     

HYDROGEN PEROXIDE MEDIATES UV-INDUCED IMPAIRMENT OF ANTIGEN PRESENTATION IN A MURINE EPIDERMAL-DERIVED DENDRITIC CELL LINE

Abstract— Ultraviolet-B (290–320 nm) radiation is known to impair the antigen-presenting cell (APC) function of Langerhans cells (LC), skin-specific members of the dendritic cell (DC) family. We sought to address mechanisms of this effect, focusing on the role played by hydrogen peroxide. For this purpose, we used a newly established murine DC line, XS52, which resembles epidermal LC in several respects. The APC capacity of XS52 cells, using two different CD4* T cell clones as responders, was inhibited significantly (>50%) by exposure to UV radiation (unfiltered FS20 sunlamps) at relatively small fluences (50–100 J/m²). Ultraviolet radiation also inhibited growth factor-dependent proliferation of XS52 cells. On the other hand, cell surface phenotype was relatively well preserved after irradiation; expression levels of B7-1 and B7-2 were reduced slightly, while other molecules ( e.g. Ia, CD54, CD1 la and CD18) were not affected. With respect to the role played by hydrogen peroxide, pretreatment with purified catalase (900 U/mL) prevented UV-induced inhibition of APC function. Short-term exposure to 3 miM H₂0₂ or f-butyl H₂0₂ mimicked UV radiation by inhibiting APC function. Finally, intrinsic catalase activity was substantially lower in XS52 cells compared with Pam 212 keratinocytes. These results indicate that the generation of hydrogen peroxide alone is sufficient to produce some, but not all, of the deleterious effects of UV radiation on DC derived from the skin.     

Modifications of in vitro skin penetration under solar irradiation: evaluation on flow-through diffusion cells

The effect of solar irradiation on ex vivo dermatomed hairless rat skin samples maintained in culture on flow-through diffusion cells for at least 24 h was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and by histological observations. Transepidermal water loss (TEWL) measurements and kinetic analysis of the permeation of both tritiated water and 14C caffeine through the skin were performed after full-spectrum solar exposure involving the use of a xenon arc solar simulator. After a UV exposure of less than 420 mJ/cm2, skin integrity and permeation of both water and caffeine did not change significantly. In contrast, after a 420 mJ/cm2 UV exposure, the epidermis appeared more contracted, associated with an increase of 55% of TEWL and 220% of the skin permeation of tritiated water after 6 h. The data suggested a dramatic alteration of the skin barrier integrity. Moreover, the flux of 14C caffeine increased rapidly by 338% of the absorption of water 12 h after irradiation. These results reveal the presence of a threshold UV exposure that would not modify skin penetration.     

THE EFFECT OF CHRONIC APPLICATIONS OF TRETINOIN(RETINOIC ACID) ON ACUTE RESPONSES OF MAMMALIAN SKIN TO UVB RADIATION IN VIVO

Abstract The effects of long—term applications of tretinoin(retinoic acid; RA) on acute responses to UVB radiation were examined in hairless mouse skin in vivo. The skin was examined histologically and the radioactive tracer [³H]-thymidine was used to study premitotic semiconservative DNA synthesis in the epidermal basal cells. Ten and 23 wk applications of a 0.05% RA solution (3 times a wk) induced epidermal acanthosis, hypertrophy of the individual cells and an increased number of [³H]-thymidine-labelled basal cells. At both time periods there was a distinct reduction in the expected inhibition of DNA synthesis at 4 h and the expected acceleration of DNA synthesis at 48 h after a single exposure to UVB radiation. In addition, the histologic morphology was unchanged at 48 h post irradiation. Thus, the repeated RA applications had a definitive influence on at least two aspects of the cutaneous response to UVB energy.     

The influence of UV exposure on 5-aminolevulinic acid-induced protoporphyrin IX production in skin.

The skin of nude mice was exposed to erythemogenic doses of UV radiation, which resulted in erythema with edema. An ointment containing 5-aminolevulinic acid (ALA) was topically applied on mouse and human skin. Differences in the kinetics of protoporphyrin accumulation were investigated in normal and UV-exposed skin. At 24 and 48 h after UV exposure, skin produced significantly less protoporphyrin IX (PpIX) than skin unexposed to UV. Human skin on body sites frequently exposed to solar radiation (the lower arm) also produced less PpIX than skin exposed more rarely to the sun (the upper arm). It is concluded that UV radiation introduces persisting changes in the skin, relevant to its capability of producing PpIX from ALA. The observed differences in ALA-induced PpIX fluorescence may be the result of altered penetration of ALA through the stratum corneum or altered metabolizing ability of normal and UV-exposed skin (or both).     

Chronic Low-Dose UVA Irradiation Induces Local Suppression of Contact Hypersensitivity, Langerhans Cell Depletion and Suppressor Cell Activation in C3H/HeJ Mice

Abstract— It has previously been demonstrated that chronic low-dose solar-simulated UV radiation could induce both local and systemic immunosuppression as well as tolerance to a topically applied hapten. In this study, we have used a chronic low-dose UV-irradiation protocol to investigate the effects of UVA on the skin immune system of C3H/HeJ mice. Irradiation with UVA+B significantly suppressed the local and systemic primary contact hypersensitivity (CHS) response to the hapten 2,4,6-trinitrochlo-robenzene. Furthermore UVA+B reduced Langerhans cell (LC) and dendritic epidermal T cell (DETC) densities in chronically UV-irradiated mice. Ultraviolet A irradiation induced local, but not systemic, immunosuppression and reduced LC (32%) but not DETC from the epidermis compared to the shaved control animals. Treatment of mice with both UVA+B and UVA radiation also induced an impaired secondary CHS response, and this tolerance was transferable with spleen cells. These results suggest that depletion of LC, but not DETC, may be involved in UVA-induced local immunosuppression in our model, and that tolerance was induced in the presence of normal numbers of DETC. Hence exposure of C3H/HeJ mice 5 days per week for 4 weeks with UVA can induce local immunosuppression and tolerance.     

INDUCTION OF SKIN TUMORS IN HAIRLESS MICE BY A SINGLE EXPOSURE TO UV RADIATION*

Abstract— Skin tumors were induced in hairless mutant mice following a single exposure to ultraviolet radiation (UV). Tumors were first noted as early as 7 weeks following irradiation. The UV, emitted by FS20/40T12 fluorescent lamps, was principally in the 280–320 nm spectral region with a peak at 313 nm. Single (skin surface) doses of 3 times 10⁴ J/m² to 24 times 10⁴ J/m² were delivered in 3 h or less. The higher doses resulted in more severe acute damage as well as greater tumor yield. Most of the tumors were benign hyperplastic epithelial papillomas; 4 out of 96 tumors examined histologically proved to be squamous cell carcinomas. This appears to be the first report of experimental carcinogenesis due to a single UV exposure, not requiring exogenous chemical promotion.     

ULTRAVIOLET RADIATION INDUCED CHANGES IN MACROPHAGE ACTIVITY

Abstract— The effect of ultraviolet (UV) radiation on macrophage activity was examined. Thioglycollate-stimulated peritoneal exudate cells were collected from adult C57BL/6 mice. Ninety-five per cent of the cells adhering to plastic petri dishes were macrophages as determined by the presence of a non-specific esterase. Adherent cells were exposed to UV radiation of 0.5-13.2 J/m². Viability and phagocytosis were measured at 0, 24, 48, 72 and 96 h after exposure. A statistically significant UV exposure-dependent decrease in macrophage viability and phagocytic capacity was observed. Macrophage viability and phagocytosis also decreased as a function of time after exposure to UV radiation.     

Proteomics Analysis of T Lymphocytes Damage Induced by Ionizing Irradiation

Human T lymphocytes were found to be highly radiosensitive and complex cellular responses including apoptosis could be induced upon exposure to X‐ray irradiation. However, the mechanism of apoptosis associated with irradiation was not clear. In this study, a proteomic method was applied to investigation on alteration of proteome of human T‐lymphocyte cells after irradiation. The Jurkat cells were irradiated with 4 Gy X‐ray and the cell lysates were collected at different times after irradiation (6, 12, 18, 24 and 48 h). The whole proteins were separated and quantified by two‐dimensional fluorescence difference gel electrophoresis, and then the differentially expressed proteins were identified by mass spectrometry. 4 proteins exhibited significant irradiation‐induced difference in abundance, including L‐plastin, bifunctional purine biosynthesis protein, tubulin beta chain, beta‐actin. Differentially expressed proteins were reported to be directly or indirectly involved in the function of human T lymphocyte. Thus, this study might provide clues to identify proteins with biological significance related to irradiation.     

Ultraviolet radiation exposure, vitamin D, and cancer

This paper reviews briefly the evidence for an association between various measures of UV radiation (UVR) exposure and the development of cancer. Issues such as data quality, study design, measurement variation, comparability of studies, and quantification of UV exposure in relation to skin cancer are discussed. A range of exposure, based on skin type, might be appropriate: from 5 min a day three times a week for light-skinned individuals and 10 min a day three times a week for darker-skinned individuals. These exposures translate into 13 h per year, for a light-skinned individual, leading to 650 h of exposure from birth to age 50.     

THE EFFECT OF ULTRAVIOLET LIGHT ON THE CYCLIC NUCLEOTIDE SYSTEM OF HUMAN FIBROBLASTS

Abstract— The concentrations of cyclic AMP and cyclic GMP in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m², and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, our results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may affect the extranuclear functions of irradiated cells.     

Effects of Ultraviolet A-1 Radiation on Calcineurin Activity and Cytokine Production in (Skin) Cell Cultures†

Calcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has been shown to positively influence various inflammatory skin diseases. Recently, Cn activity has been demonstrated in skin and skin cell cultures. In the present study we have investigated the effects of UV(A-1) irradiation on Cn activity in skin. In total skin we found a significant reduction in Cn activity after exposure to 450 kJ m⁻² of UVA-1 (340–400 nm). In repeated experiments cultures of fibroblasts and keratinocytes also showed dose-dependent and selective reduction in Cn activity after UVA-1 irradiation. UVB irradiation caused a decrease in the Cn activity of one of two fibroblast cultures and was ineffective in keratinocytes. In Jurkat cells and PBMC UVA-1 reduced Cn activity and also the production of cytokines such as interleukin (IL)-2, γ-interferon, IL-4 and IL-10 that are controlled by the Ca²⁺–Cn pathway. These results indicate that UV(A-1) irradiation may lead to inactivation of Cn in the skin and thus suppress the skin immune system in a similar fashion to the Cn inhibitors.     

Sunscreens Protect from UV-Promoted Squamous Cell Carcinoma in Mice Chronically Irradiated with Doses of UV Radiation Insufficient to Cause Edema

Previously we reported that the broad-spectrum sunscreen microfine titanium dioxide (MTD) could completely protect C3H/HeJ mice from UV radiation-induced immunosuppression to a contact sensitizer. In contrast, 2-ethylhexyl p-methoxycinnamate (2-EHMC), a UVB-absorbing sunscreen, only partially protected the skin immune system. In this study we investigated further this differential protection of the skin immune system by comparing the ability of 2-EHMC and MTD to protect these mice from the promotion phase of tumorigenesis. The mice were initiated using a single subcarcinogenic dose of 7,12-dimethylbenz(a)anthracene (DMBA) followed by promotion with chronic low-dose solar-simulated UV radiation for 32 weeks. We used doses of UV insufficient to cause edema in order to simulate daily human exposure to solar UV radiation. Mice were observed for the appearance of squamous cell carcinomas for 48 weeks. The DMBA-initiation alone and DMBA-initiated, sunscreen-treated groups did not develop tumors. Ultraviolet alone induced the appearance of tumors in 46% of mice at week 48 and therefore some tumors were initiated by UV. Initiation with DMBA prior to UV irradiation enhanced tumorigenesis such that 87% of mice at week 48 had tumors. Both 2-EHMC and MTD completely protected these mice from UV-induced promotion as well as from complete carcinogenesis despite the different UV-absorption spectra of the sunscreens and their differential abilities to protect from UV-induced immunosuppression. Furthermore, we have shown that, if UV exposure is not increased to compensate for tolerance to edema, protection from tumorigenesis is afforded by sunscreens.