Vascular changes in human skin after ultraviolet irradiation

Blood-flow changes in human skin after ultraviolet irradiation at 250 and 300 nm have been measured by three separate methods. Those methods which measure overall blood-flow changes in the skin showed increased flow after irradiation at both wavelengths. A method which measured flow only in the superficial vessels showed a slight increase in flow after low doses of both wavelengths, but in contrast, after higher doses, this method showed a marked reduction in flow through the upper dermal vessels. This reduction in flow is probably due to stasis in these superficial vessels, perhaps secondary to vascular damage. Contrary to previous reports, blood-flow changes after irradiation at 250 and 300 nm are similar and may be mediated by identical mechanisms.     

Mitochondrial dysfunction and cellular stress progression after ultraviolet B irradiation in human keratinocytes

Background: Ultraviolet (UV) radiation is the major environmental harmful factor that affects human skin. UVB radiation is known to be a potent inducer of reactive oxygen species (ROS) production and has also been associated with the generation of nitric oxide (NO), all of which have been implicated in various skin disorders. It is well known that mitochondria can also be affected by UVB, leading to alterations in their membrane structure and permeabilization with cytochrome c release, which consequently affects the cell function. However, the loss of keratinocyte mitochondrial function generated by UVB, as well as its kinetics, has not been characterized completely.
Methods: We evaluated the effect of UVB irradiation on HaCat cells' mitochondrial function, assessed by membrane potential loss and superoxide anion (O₂^•−) production, correlating with apoptosis, p53 expression, ROS levels and NO production, 0, 6, 12, 24 and 48 h post-irradiation.
Results: HaCat cells progressed toward apoptotic cell death as the time post-irradiation increased, with the highest levels found 48 h after irradiation. Increased levels of ROS were observed 6 h after irradiation while high O₂^•− levels and mitochondrial membrane depolarization were detected 12 h post-UVB. Nevertheless, NO production was not significantly increased at any of the evaluated times.
Conclusions: The kinetics of mitochondrial dysfunction after UVB irradiation in human keratinocytes progressed in a time post-irradiation-dependent manner, and they are closely related to cell death. However, there are certain levels of apoptosis, although low, in the absence of mitochondrial alterations. In addition, our data suggest that ROS play a greater role in keratinocyte UVB damage than reactive nitrogen species.     

The expression of melanogenic proteins in Korean skin after ultraviolet irradiation

For proper melanin production, several specific enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase are required. Their expressions are increased after exposure to UVB. However, it is not known how long tyrosinase and TRP-1 activities continue after UV irradiation in vivo. The purpose of this study is to measure the changes in expressions of tyrosinase, TRP1, and MITF after exposure to UV on skin in a Korean population. We established an immunohistochemical staining protocol for specimens which were obtained from UV-irradiated skin in five healthy Korean males on the 2nd, 5th, 7th, 28th, and 56th days after UV irradiation. Tyrosinase, TRP-1, and MITF expressions increased until 7 days after UV irradiation and then dropped to the basal constitutive level 4 and 8 weeks later. Interestingly, tyrosinase increased prior to TRP-1. This study reveals the time-sequence of melanin-synthesized enzymes and provides important information for the clinical evaluation of the effectiveness of whitening agents.     

Sunburn cell formation, dendritic cell migration, and immunomodulatory factor production after solar-simulated irradiation of sunscreen-treated human skin explants in vitro

Using human skin explants, we investigated the effects of two different sunscreen preparations containing a chemical UVB filter alone [sun protection factor (SPF) 5.2] or UVA+UVB filter [SPF 6.2] on sunburn cell formation, dendritic cell (DC) migration, CD86- and CD1a-positive cell number, and tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-1, IL-10, and IL-12 production in the skin after irradiation with different doses of solar-simulated UV radiation. Sunscreen- or placebo-treated skin explants were irradiated with solar-simulated UV radiation at 0.5, 1, and 2 minimal erythematous dose equivalents (MEDE) (as determined in an in vivo human study) multiplied by the SPF of the placebo or sunscreens. After irradiation, skin explants were floated on RMPI medium for 48 h. Cells that had emigrated and the skin explants were histologically analyzed, and the soluble mediators were measured in the supernatants by ELISA. Exposure to UV radiation led to concentration-dependent increases in sunburn cell formation and TNFalpha production but a concentration-dependent decrease in DC migration and CD86- and CD1a-positive cell number in the epidermis. Both chemical sunscreens protected against those alterations. The immunoprotective capacity of the sunscreens correlated with their SPF but was independent of the sunscreens' UVA protection capacity, suggesting that UVA is not a major factor for immunosuppression under the conditions used in the model. UV irradiation did not significantly affect the vitality of emigrated DC; the expression of HLA, CD80, and lag on emigrated cells; the number of CD1a-positive cells in the dermis; or the production of IL-1, IL-10, and IL-12. We conclude that our model may be useful in determining the immunoprotective capacity of sunscreens.     

Effect of ultraviolet adaptation on the ultraviolet absorption spectra of human skin in vivo

Background: The absorption spectrum of human skin provides a basis for the estimation of the possible photobiological impact of ultraviolet (UV) radiation. The optical properties of human skin in the UV spectral range have so far mainly been measured ex vivo due to a lack of an appropriate in vivo technique and the change of optical properties during the course of adaptation to higher UV doses has hardly been addressed. Methods: We have determined the absorption spectra of human skin in vivo in the wavelength range from 290 to 341 nm in 3 nm steps using laser optoacoustics. In this technique, optical properties are derived from the pressure profile generated by absorbed light energy in the sample. Spectra from the volar and dorsal aspects of the forearm of 20 subjects were compared, i.e. sites with native and various facultative pigmentation. Results: UV adaptation shows as an increase in absorption coefficients over the entire measured UV range and especially in short‐range UVB. Subject groups with high vs. low UV exposure can be discriminated by analyzing the difference absorption spectra between dorsal and volar aspects of the forearm. No dependence on the subject's phototype was seen in the degree of adaptation. Conclusion: The difference between native and facultative pigmentation may be explained by the absorption properties of the two prime chromophores responsible for adaptation to higher UV exposure: melanin and keratin. Stronger pigmentation, i.e. a higher melanin concentration, is found as an increase of absorption coefficients over the entire UVA‐II/UVB range. The thickening of the horny layer and accordingly, a higher influence of keratin on the absorption spectra is prominent especially in the UVB region.     

Epidermal changes in human skin following irradiation with either UVB or UVA

We have demonstrated previously that following UVB irradiation to normal volunteers there is an increase in epidermal and stratum corneum thickness and an increase in the thymidine autoradiographic labeling index. These changes are coupled with alterations in epidermal glucose-6-phosphate dehydrogenase and succinic dehydrogenase activities, despite the absence of erythema clinically. The use of a sunscreen did not completely prevent these changes. In this study, we have examined the effects of repeated irradiation of human skin with either UVB or UVA alone in order to compare the changes produced in the epidermis and to ascertain whether UVA irradiation could cause these. Irradiation with either UVB or UVA alone was found to increase the mean epidermal thickness, the mean stratum corneum thickness, and mean keratinocyte height significantly. Glucose-6-phosphate dehydrogenase activity was significantly increased throughout the epidermis, and succinic dehydrogenase activity was significantly decreased. The autoradiographic labeling index was significantly increased following UVB irradiation but not following UVA irradiation. These results demonstrate that UVA alone can have a direct effect on epidermal morphology and metabolism, suggesting that protection of skin from UV radiation should include adequate protection from UVA.     

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.     

Mitochondrial function evaluation in epidermal cells ex vivo after ultraviolet irradiation

Ultraviolet radiation (UVR) effects on skin have been extensively studied. However, mitochondrial dysfunction and superoxide () production have only been studied using cell cultures, which are useful models, but do not consider the crosstalk between tissues or cellular differentiation. We aimed to evaluate the usefulness of fluorescent dyes to study skin ex vivo. Mitochondrial alterations were evaluated in epidermal cells isolated from UVR-exposed mice. Furthermore, a combination of dyes and antibodies was tested to analyse specific skin cell types. UVR caused a decrease in the percentage of total cells with polarized mitochondria, but did not change the mitochondrial production. However, this production was increased significantly in cells. Furthermore, it was possible to evaluate the cellular damage produced to basal keratinocytes and Langerhans cells. The results show that fluorescent dyes - alone or in combination with antibodies - are useful to analyse cellular events that take place in whole organs.     

Changes in the skin moisture contents, skin color, and skin protein conformational structures of sprague-dawley rats after ultraviolet B irradiation.

The influence of ultraviolet B (UVB) irradiation on the skin moisture contents, skin color, and protein secondary structure in the skin of alive Sprague-Dawley (SD) rats was investigated. The UVB irradiation source emitted 725 +/- 10 microW/cm(2) of UVB range with a peak at 302 nm during the exposure course of 6 h/day for 3 successive days. The moisture content of SD rat's skin first decreased with the UVB irradiation, then returned to normal level on days 1 and 2, and, finally, decreased significantly with the increase of UVB irradiation on day 3. After the 6-hour irradiation on days 1 and 2, the moisture contents of the irradiated skin of SD rat returned to normal values, suggesting repairability of the irradiated skin of SD rat during the following 18 hour period without irradiation. By increasing frequency and duration of UVB irradiation, the skin color parameters exhibited a lower L* value, a greater a* value, and a higher DeltaE value, indicating that the skin color darkened and reddened. The slight modification of protein secondary structure of the skin of SD rats after repeated UVB irradiation was evidenced. However, the infrared spectrum during the initial course of irradiation, from day 1 to day 3, exhibited a similar pattern. Thus, the alive SD rat's skin was repairable during the 18-hour period without irradiation.     

In vivo ultraviolet irradiation of human skin results in profound perturbation of the immune system. Relevance to ultraviolet-induced skin cancer.

Ultraviolet exposure of human skin deletes the function of antigen-presenting Langerhans cells normally resident within the epidermis. Langerhans cells are capable of activating T-lymphocytes by presenting antigens (such as nickel or tumor antigens) to T-lymphocytes. Such activated T-lymphocytes may be involved in the development of contact dermatitis and the immune surveillance of immunogenic skin cancers. Deletion of the function of Langerhans cells does not result in abrogated epidermal antigen presentation since ultraviolet irradiation simultaneously induces the appearance of another epidermal antigen-presenting cell population that is distinct from the Langerhans cell population and seems to induce suppression of the immune response. Suppression of the immune response following ultraviolet irradiation in murine models is critical for growth of immunogenic ultraviolet-induced skin neoplasm. Thus, ultraviolet irradiation of human skin may facilitate the growth of human neoplasms, and the spreading of skin-associated infections due to induction of suppressor T cells.     

iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell precursors: a mechanism for transiently decreased dendritic cells in vivo after human skin injury by ultraviolet B

Our previous data indicated that C3, its bioactive product iC3b, and the iC3b ligand CD11b are critical for ultraviolet-induced immunosuppression. We thus hypothesized that iC3b is an important skin-based factor regulating CD11b+ monocytic cell function in the acute post-ultraviolet period. Although monocytic cell migration peaked at 1-3 d after ultraviolet exposure of skin, dermal CD1c dendritic cells underwent a rapid and prolonged depletion that did not recover until day 7. Because ultraviolet-induced iC3b deposits are reciprocally maximal on day 3, but fade by day 7, we next hypothesized that iC3b can be responsible for the delay in differentiation into dendritic cells of monocytic cells migrating into ultraviolet-exposed skin. Analysis of dermal cells derived from keratome biopsies suggested that iC3b exposure could inhibit the development of CD1c+ dermal cells. To model newly immigrating blood monocytes entering ultraviolet-exposed, iC3b-containing dermis, purified monocytes from human blood were induced with granulocyte-macrophage colony stimulating factor to generate a population of dendritic cell precursors expressing CD1c. Incubation with iC3b markedly inhibited the appearance of CD1c+ cells (p<0.05) and induced CD1c-CD14+ cells. This inhibition was reversed by coincubation with an anti-CD11b antibody that blocks the iC3b binding site. Other functions associated with dendritic cell maturation were also inhibited by iC3b, such as interleukin-12p70 production as well as CD80 and CD40 expression. Restimulation of monocytes for DC maturation revealed that iC3b induced a temporary inhibition of DC differentiation. Thus, a human skin response in which iC3b is transiently (3-7 d) generated in dermis, such as ultraviolet, can arrest monocytic skin-infiltrating cells from undergoing dendritic cell precursor differentiation.