Effects of 8-Methoxypsoralen (8-MOP) and UVA on human lymphocytes

Summary Peripheral lymphocytes of 37 psoriatic patients are tested before and under PUVA treatment using as parameter the non specific stimulation effect of HgCl₂ (10 µg/ml) in culture, measuring the³H-thymidine incorporation after the last 16 h of a 5-days culture. Oral 8-MOP in therapeutic doses is decreasing the lymphocyte stimulation as well as 8-MOP together with UVA irradiation during the first week of treatment. After 1 week, the stimulation is, on the contrary, significantly enhanced after irradiation. Lymphocytes isolated by centrifugation over Lymphoprep are submitted to PUVA conditions in petri dishes (Hank's solution 8-MOP 1 µg/ml, irradiation with 350 nm, 93-372 mJ/cm²). The total cell number, the E-rosette formation (as marker for T-Lymphocytes) and the EAC-rosette formation (as marker for B-Lymphocytes) are determined. PUVA conditions have an energy dependent decreasing effect on the cell number, while the T- and B-cell proportions remain constant. UVA irradiation alone has such an effect only with high energies. 8-MOP without UVA has no significant influence on the cell number.Offprint requests to: Dr. G. Lischka (address see above)     

Comparison of the effect of 8-methoxypsoralen (8-MOP) plus UVA (PUVA) on human melanocytes in vitiligo vulgaris and in vitro.

In this study, we examined the various effects of PUVA treatment on cultured human melanocytes, and it revealed that 1) the higher the dose of PUVA treatment, the more significant the inhibition of cell DNA and protein synthesis; 2) the higher the dose of PUVA treatment, the more significant the depletion of epidermal growth factor receptor (EGFR) expression; 3) PUVA treatment at 124 mJoule/cm2 depleted the vitiligo-associated melanocyte antigens (VAMA) immediately after irradiation, and both the VAMA and EGFR expression progressively recovered at 24 or 72 h after PUVA; 4) PUVA treatment stimulated tyrosinase activity, but not in a dose-dependent fashion. In vitiligo vulgaris, PUVA treatment may stimulate the regrowth of melanocytes from hair follicles, but deplete the epidermal Langerhans cells in depigmented lesion of patients with stable vitiligo. Comparing the above results obtained from in vivo and in vitro studies, it reveals significantly different biologic responses. Although the precise therapeutic mechanism of PUVA treatment in vitiligo is still not well known, it is proposed that 1) PUVA treatment may stimulate the other components of skin, such as keratinocytes, to release inflammatory mediators and some of them may act as melanocyte growth-stimulatory factors (MGSF), which further enhance the proliferation of remaining melanocytes in hair follicle; and 2) PUVA treatment may deplete the VAMA expression on cell membrane of melanocytes and also deplete epidermal Langerhans cells, which may result in blocking the progressing of antibody-dependent cell-mediated cytotoxicity to melanocytes in vitiligo.     

Evaluation of time-dependent response to psoralen plus UVA (PUVA) treatment with topical 8-methoxypsoralen (8-MOP) gel in palmoplantar dermatoses

BACKGROUND: Topical psoralen plus UVA (PUVA) is an effective treatment for localized forms of eczema, psoriasis, and palmoplantar pustulosis, which avoids some of the undesirable side-effects of systemic psoralens. Aims In this study, the efficacy of topical PUVA treatment with 8-methoxypsoralen (8-MOP) gel was compared with placebo plus UVA in chronic recurrent palmoplantar dermatoses. METHODS: Twenty-two patients with palmoplantar disease (11 with psoriasis vulgaris, six with eczema, and five with pustulosis) were enrolled in the study. The study design was a left-right comparison: one hand or foot was treated with 8-MOP 0.01% gel plus UVA, whilst the contralateral hand or foot received placebo and UVA for 6 weeks. Twenty minutes after application of the gel, both sides were exposed to UVA. The treatment regimen was three times a week, and the UVA dose was increased weekly by 20%. RESULTS: A comparison of the pre- and post-treatment scores with regard to the severity of the clinical picture and the infiltration of plaques showed a significant decrease (from 7.5 +/- 2.0 to 2.5 +/- 2.1 and from 2.0 +/- 0.7 to 0.3 +/- 0.5, respectively) in the sites treated with 8-MOP gel compared with placebo after 6 weeks. CONCLUSION: The results of the study indicate that at least 18 courses of local PUVA within 6 weeks, with a cumulative dose of 87 J/cm(2), are required to induce a significant decrease in the disease severity and an improvement in the infiltration of plaques due to 8-MOP gel at a concentration of 0.01% when treating chronic recurrent palmoplantar dermatoses.     

Photoprotective effect of calcipotriol upon skin photoreaction to UVA and UVB

It has been shown that 1,25-dihydroxyvitamin D₃ has a photoprotective effect against UVB injury in mouse skin and cultured rat keratinocytes by induction of metallothionein (MT). Calcipotriol is a synthetic analogue of 1,25-dihydroxyvitamin D₃ with equipotent cell regulating properties, but with a lower risk of calcium-related side effects. The aim of the present study was to see whether calcipotriol has a photoprotective property both in vitro and in vivo. We examined the effect of calcipotriol on UV-induced damage of cultured human keratinocytes through a cell viability assay, and measurement of DNA synthesis by cultured keratinocytes, on UV-induced damage of mouse skin and on minimal erythema dose (MED). We found that calcipotriol was protective against UVB-induced reduction in DNA synthetic activity of cultured keratinocytes in relatively low doses (20 and 40 mJ/cm²) of UVB. With phototesting following application of calcipotriol, five subjects among 10 healthy volunteers and three among six psoriasis patients showed an increase in MED compared with the vehicle-treated site. These findings imply that calcipotriol may be photoprotective and that more extensive studies with various doses of UV irradiation and modes of calcipotriol delivery are required.     

Correlation between bathing time and photosensitivity in 8-methoxypsoralen (8-MOP) bath PUVA

Bath PUVA (psoralen plus ultraviolet A) using 8-methoxypsoralen has become increasingly popular in recent years as an effective treatment option for a continuously expanding range of skin disorders. Among the various variables of bath PUVA treatment, the impact of bathing time on photosensitivity has never been investigated in detail. We therefore determined the threshold UVA dose for erythema induction after different bathing periods. A marked influence of bathing time on photosensitivity was found. Increasing the soaking period from 5 min to 30 min resulted in a greater than 60% reduction of the minimal phototoxic and minimal perceptible phototoxic dose. Our results demonstrate that the duration of the psoralen bath is a critical parameter in bath PUVA treatment and has a major influence on UVA dose requirements.     

Protection against pyrimidine dimers, p53, and 8-hydroxy-2'-deoxyguanosine expression in ultraviolet-irradiated human skin by sunscreens: difference between UVB + UVA and UVB alone sunscreens

As DNA damage induced by ultraviolet radiation plays an essential role in skin cancer induction, we pursued the measure of several DNA lesions induced by ultraviolet radiation in human skin for determining the efficacy of different topical photoprotectors. Non-exposed skin (buttocks from 20 individuals) was exposed to 10 doses of ultraviolet, which corresponded to three to four minimal erythema doses of solar-simulating radiation, and biopsies were taken at 24 h within the half and one minimal erythema dose sites and a nonirradiated, adjacent control area. We report that even suberythemal doses of ultraviolet radiation are capable of inducing substantial DNA damage, namely pyrimidine dimers, p53 induction, and the DNA base-modified product generated by oxidative stress, 8-hydroxy-2'-deoxyguanosine. All three lesions are induced in a dose-dependent manner. An additional eight individuals were treated with either ultraviolet B or ultraviolet B + ultraviolet A sunblock (sun protection factor 15) and exposed to 71/2 and 15 times the minimal erythema dose on each individual, with biopsies taken at 24 h post-ultraviolet. Pyrimidine dimer and p53 expression were rarely seen in nonirradiated skin but occasional staining was seen in all normal skin for 8-hydroxy-2'-deoxyguanosine. Applications of sunscreens to human skin before irradiation were shown to attenuate erythema but did not completely eliminate all three types of cellular damage when tested up to their sun protection factor 15. Furthermore, ultraviolet B + ultraviolet A sunscreens were less efficient than the ultraviolet B alone formulation for protection against all three lesions. These results suggest that DNA damage assessed in vivo by immunohistochemistry provides a very sensitive endpoint for determining the efficacy or photosensitivity of possible different protective measures in human skin.     

Short- and long-wave UV light (UVB and UVA) induce similar mutations in human skin cells

While the mutagenic and carcinogenic properties of longwave UV light (UVA) are well established, mechanisms of UVA mutagenesis remain a matter of debate. To elucidate the mechanisms of mutation formation with UVA in human skin, we determined the spectra of UVA- and UVB-induced mutations in primary human fibroblasts. As with UVB, we found the majority of mutations to be C-to-T transitions also with UVA. For both UVA and UVB, these transitions were found within runs of pyrimidines, at identical hotspots, and with the same predilection for the nontranscribed strand. They also included CC-to-TT tandem mutations. Therefore, these mutations point to a major role of pyrimidine dimers not only in UVB but also in UVA mutagenesis. While some differences were noted, the similarity between the spectra of UVA- and UVB-induced mutations further supports similar mechanisms of mutation formation. A non-dimer type of DNA damage does not appear to play a major role in either UVA or UVB mutagenesis. Therefore, the previously reported increasing mutagenicity per dimer with increasing wavelengths cannot be due to non-dimer DNA damage. Differences in the cellular response to UVA and UVB, such as the less prominent activation of p53 by UVA, might determine a different mutagenic outcome of UVA- and UVB-induced dimers.     

Effect of 8-methoxypsoralen plus UVA (PUVA) on lymphocyte transformation and T cells in psoriatic patients

The peripheral blood lympochytes of the psoriatic patients studied initially showed a normal response to phytohaemagglutinin (PHA) and to purified protein derivative of turberculin (PPD) in a microculture using whole blood, but a lower than normal response to a high concentration of concanavalin A (Con A). The initial level of E-rosette-forming T cells in psoriatic patients (51.3%) was significantly lower than that in healthy controls (66.7%). A single exposure to 8-methoxypsoralen (8-MOP) and UVA light (PUVA) did not suppress the lymphocyte responses to PHA, Con A or PPD, rather the responses showed a tendency to be slightly increased. Similarly, no changes in the mitogen responses of peripheral blood lymphocytes were recorded during or after 12 weeks PUVA therapy for psoriasis. The lymphocytes still showed a weaker than normal response to a high concentration of Con A. However, the percentage of E-rosette-forming T cells in the peripheral blood increased from 51.3% to 62.8% after 12 weeks PUVA therapy. The low initial level of E-rosette-forming T cells was found to correlate more closely with the activity than with the extent of the disease. The increase found in the E-rosette-forming cells during PUVA therapy did not correlate with the improvement of the psoriatic lesions. The low response of peripheral blood lymphocytes of psoriatic patients to a high concentration of Con A correlated with the age of the patients but not with the activity or extent of the disease.     

Effect of antioxidants and free radical scavengers on protection of human skin against UVB, UVA and IR irradiation

Background/aims: This study was designed to observe the effect of a mix of commercial antioxidants and free radical scavengers (AO) on protection of human skin against different wavelengths of the solar spectrum: 280-320 nm (UVB), 320-400 nm (UVA) and 400-900 nm (IR).
Methods: Healthy volunteers were chosen from the local population and exposed to the three aspects of the solar spectrum. Erythema at 24 h, immediate pigment darkening (IPD) and immediate erythema were employed as markers for UVB, UVA and IR exposure, respectively.
Results: Based on 24 h erythema measurements, the AO mix afforded about 2.6-fold (163%) protection against UVB-induced erythema (Table 1). IPD measurements after UVA exposure exhibited 76% protection afforded by the AO mix. The AO mix reduced IR-induced increases in erythema and blood flow by 43% and 52%, respectively (Table 1).
Conclusions: Data from these studies suggest that treatment with antioxidants prior to exposure to solar irradiation allows protection of human skin against IR as well as UVB and UVA. Antioxidants and free radical scavengers may be valuable adjuncts to traditional sunscreens, for protection of skin against the effects of actinic exposure that, over a long term, can result in formidable consequences such as skin cancer and photo-aging.     

不同波长紫外线照射后皮肤颜色的变化

目的 观察不同波长紫外线照射皮肤后，颜色变化的过程。方法 用双倍剂量最小持续性黑化量和最小红斑量对10例Ⅲ型受试者皮肤进行照射，通过临床评分、扫描反射比分光光度仪和窄谱反射分光光度计三种方法对照射后的皮肤进行14天的评价和测定。结果 UVB照射后，a*值和红斑指数（EI值）在照射后6 h急剧增加，照射后2天达到高峰；L*值在照射后1天出现急剧降低；ITA°在第7天显著降低；黑素指数（MI值）在照射后2天内有逆向的降低趋势，直到照射后7天才有显著增高。在UVA照射下，a*值和EI值改变不明显；L*值在照射后6 h出现显著降低；ITA°在第14天达到最低值； MI值仅照射后1天有显著增高。结论 UVA和UVB照射后的皮肤颜色改变在时间动力学和反应程度方面有明显区别。a*值和EI值是评价照射后日晒伤较为敏感而准确的参数，而ITA°和MI值是评价晒斑较好的参数。     

Low doses of UVB or UVA induce chromosomal aberrations in cultured human skin cells

Chromosomal defects are frequently present in malignant and premalignant skin disorders; however, it is not known whether ultraviolet radiation from sunlight plays a role in their induction. To obtain information on the ability of ultraviolet A and ultraviolet B to induce chromosomal aberrations, cultured melanocytes and fibroblasts were exposed to physiologic doses of ultraviolet A or ultraviolet B and, for comparison, to gamma rays. As a measure of chromosomal aberrations, the formation of micronuclei was determined. To obtain sufficient statistical data on induced micronuclei and cell kinetics, a flow cytometry method has been modified and applied. The flow cytometry method analysis is based on staining the DNA with ethidium bromide and the cell membranes with 1,6-diphenyl-1,3,5,-hexatriene. We observed dose-dependent micronuclei formation after gamma or ultraviolet B irradiation in both cell types and also for ultraviolet A in fibroblasts. The yield of micronuclei induced in fibroblasts by ultraviolet A was only a factor 15 smaller than that induced by ultraviolet B (313 nm). The results indicate that 10 kJ per m2 (equivalent to 1 minimal erythema dose) of ultraviolet B and 150 kJ per m2 of ultraviolet A (0.2 minimal erythema dose) can induce 1% of micronuclei in fibroblasts, equivalent to the induction due to 0.6 Gy of gamma radiation. In conclusion, physiologic doses of sunlight can induce chromosomal aberrations at a level comparable with that observed after exposure to approximately 1 Gy of ionizing radiation. Therefore, sunlight can be considered a potential inducer of chromosomal aberrations in skin cells, which may contribute to skin carcinogenesis.     

Comparison of broadband UVB, narrowband UVB, broadband UVA and UVA1 on activation of apoptotic pathways in human peripheral blood mononuclear cells

BACKGROUND/PURPOSE: Ultraviolet (UV) radiation is an important therapy for immune-mediated cutaneous diseases. Activation of early apoptotic pathways may play a role in the clinical effectiveness. Different UV wavelengths have different efficacy for various diseases, but it remains unclear whether the ability to induce apoptosis differs with respect to the wavelength, and whether they induce apoptosis through the same mechanism. The aim of this study is to analyze the effects of different UV wavelengths that are used clinically on normal human peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were treated with UV-light sources broadband UVB, narrowband UVB, broadband UVA and UVA1. Initiation of apoptosis was assessed by flow cytometry by staining-treated cells for activated caspases. Immunoblots were performed to measure for cleaved caspase-3, -8, -9, cytochrome c, Bcl 2-interacting domain and poly-(ADP ribose) polymerase cleavage. RESULTS: We demonstrate that all the UV radiation sources induced caspase activation in a dose-and time-dependent manner. Components of both the extrinsic and intrinsic pathways of apoptosis were activated by all of the UV wavelengths tested, but differed in the level of energy needed for activation. CONCLUSION: The greater effectiveness of UVB on initiation of apoptotic pathway suggests that apoptosis may play a role in the clinical efficacy of UVB-responsive inflammatory cutaneous diseases.     

Inhibitory effect of 8-methoxysporalen plus UVA (PUVA) on the systemic induction of contact sensitivity to dinitrochlorbenzene (DNCB) in guinea pigs

Summary To investigate the influence of 8-MOP and UVA on the induction of cell-mediated immunity, guinea pigs, sensitized with a single injection of dinitrofluorbenzene (DNFB) in Freund's complete adjuvant (FCA) in all footpads and the nuchal skin, were treated with 8-MOP, UVA, or 8-MOP+ UVA on days 0, 2, and 5 and tested with dinitrochlorbenzene (DNCB) on day 14 after sensitization. Two control groups, exposed in a covered condition to the PUVA 4000 lamp, to observe the heat effect, showed a slightly enhanced contact sensitivity in comparison to the only sensitized control group. No altered reactivity was observed after irradiation with longwave UV light alone, whereas a statistically significant enhanced resp. reduced contact sensitivity was obtained after the treatment with 8-MOP alone and the combined action of 8-MOP +UVA, respectively.Dedicated to Prof. Dr. P. Laugier, Chairman of the Department of Dermatology, University of Geneva, Switzerland, on the occasion of his 70th anniversary     

CD8+ lineage dendritic cells determine adaptive immune responses to inflammasome activation upon sterile skin injury

The molecular links between sterile inflammation and induction of adaptive immunity have not been fully identified. Here, we examine how damage‐associated molecular patterns (DAMPs), as opposed to pathogen‐associated molecules (PAMPs), regulate the immune response to non–self‐antigens presented at the site of a physical injury. Heat applied briefly to the skin invokes sterile inflammation, characterized by local cell death and caspase‐1 activation without demonstrably disrupting skin integrity. Co‐delivery of ovalbumin (OVA) with heat injury induces OVA‐specific CD8⁺ T‐cell responses, and this is dependent on caspase‐1 activation and MyD88 signalling. Using Id2flox/flox‐CD11cCre+ mice, we demonstrate that CD8⁺ lineage DCs are required to induce OVA‐specific CD8⁺ T‐cell responses following heat injury. Consistent with this observation, intradermal administration of CD8⁺ lineage DCs but not CD11b⁺ lineage DCs restores priming of CD8⁺ T‐cell responses in Casp‐1^?/? mice. Thus, we conclude that a sterile injury induces CD8⁺ T‐cell immune responses to local antigen through caspase‐1 activation and requires CD8⁺ lineage DCs, a finding of significance for immunotherapy and for the pathogenesis of autoimmunity.