Estimation of the effect of increasing UVB exposure on the human immune system and related resistance to infectious diseases and tumours.

Exposure to UV light has, besides some beneficial effects (vitamin D production), many harmful effects on human health. UVB irradiation has been shown to suppress both systemic and local immune responses to a variety of antigens, including some microorganisms. However, it is still not known whether such immunomodulating effects may lead to an increase in the number and severity of certain tumours and/or infections in humans. We report herein the data provided by a project that was funded by the European Union (Programme Environment), and that was aimed at the estimation of the risk associated with increased UVB exposure due to ozone depletion regarding the deleterious effects on the immune system and related resistance to tumours and infections in humans. The data, obtained by the different research groups involved, were assembled and used to calculate for the first time a risk assessment for increased environmental exposure to UVB in human subjects.

Quercetin prevents UV-induced local immunosuppression, but does not affect UV-induced tumor growth in SKH-1 hairless mice.

Ultraviolet is thought to induce skin tumors by its dual activity as a mutagenic agent and a suppressor of cell-mediated immunity. In the present study the effects of quercetin, a flavonoid-containing compound, on carcinogenesis and immunosuppression were studied in SKH hairless mice exposed to suberythemal doses of UV for up to 17 weeks. It was found that quercetin did not affect the onset or growth of non-melanoma skin tumors resulting from UV exposure. In contrast, it prevented the suppression in contact hypersensitivity (CHS) to picryl chloride induced by UV. The mechanism of this prevention might be explained by the observation that the decreased number of epidermal Langerhans' cells is partly prevented by the quercetin. Quercetin did not alter the effects of UV in increasing numbers of spleen and lymph node cells, only partly in decreasing the CD8-positive cells in spleen cell populations and decreasing the lymphoproliferative response of spleen cells to the mitogens concanavalin A and phytohemagglutinin. Thus oral quercetin did not prevent UV-induced carcinogenesis although it restored the skin-associated CHS response probably by protecting the antigen-presenting cells in the skin.

The effect of oral quercetin on UVB-induced tumor growth and local immunosuppression in SKH-1.

The effects of quercetin (4%) on UVB-induced carcinogenesis and immunosuppression were studied in hairless SKH-1 mice exposed daily to suberythemal UVB for 12/13 and 16/17 weeks. Macroscopic and microscopic examinations showed that quercetin did not affect the onset and growth of UVB-induced non-melanoma skin tumors. Quercetin prevented the UV-induced suppression of the contact hypersensitivity (CHS) and the reduction of the percentage of CD8-positive cells in spleen and lymph nodes. Other immunological parameters were not affected. Thus, the results indicate that oral intake of a high dose of quercetin does not prevent UVB-induced carcinogenesis, although it restores the skin-associated CHS response.

Time and dose dependence of acceptance of UV-induced syngeneic tumor implants in chronically UV-exposed hairless mice.

The kinetics of skin cancer induction by UV radiation has been extensively studied in hairless mice and described by Weibull statistics in which the time till 63% of the mice bear tumors is a primary parameter. However, the kinetics of the associated immunosuppression remained to be determined. To this end, we implanted a syngeneic UV-induced skin carcinoma cell line (T51/6.53) in the ventral skin of HRA/SKH hairless mice after various periods of daily dorsal UV exposure, either 150 or 75 mJ/cm2 per day UV from F40 sunlamps (regimens that when continued yield 63% of the mice with 1 mm tumors in 11.5 or 16.2 weeks, respectively). Both exposure regimens achieved a 100% acceptance (after 7 and 16 weeks, respectively). The implants failed to grow in all unirradiated control mice, but the percentage of mice in which the implants grew increased with the UV treatment time and dose. The estimated times to 63% implant acceptance were 4.3 +/- 0.8 and 8.2 +/- 0.8 weeks for the high and low daily doses, respectively. As reported earlier for shaved haired mice, there appears to be a straight reciprocity between daily UV dose and the time to tumor acceptance, i.e. the latter fully depends on the cumulative UV dose, whereas the tumor induction does not. The latter probably also depends on a pure elapse of time, i.e. UV-independent processes. A further analysis of the Weibull description indicates that immunosuppression toward the tumor requires fewer UV-driven steps that tumor induction.

Frequent p53 alterations but low incidence of ras mutations in UV-B-induced skin tumors of hairless mice.

We have investigated UV-B-induced skin tumors of hairless SKH-HRA mice for alterations in the p53 gene and for mutations in either of the three ras genes. Out of 32 tumors screened, only one contained a ras mutation, i.e. in codon 12 of the K-ras gene. Alterations in the p53 gene were much more abundant, as illustrated immunohistochemically by the accumulation of p53 protein in 75% of the tumor sections examined. Immunoreactivity was observed primarily in the proliferative cell compartment, but no clear correlation between p53 staining in tumor cells and histological parameters for malignancy was observed. Subsequent sequence analysis showed that point mutations in the p53 gene are detectable in 30% (nine out of 30) of the skin tumors examined. The majority of the mutations are located in codons 267 and 272, most likely originating from UV-B-induced photo-adducts at dipyrimidine sites in the non-transcribed strand. Codon 272 corresponds to the human codon 278, which is also a hotspot for p53 mutations in human non-melanoma skin cancers. Codon 267 matches the human codon 273, which does not contain a dipyrimidine site, but represents a CpG hotspot for p53 mutations in internal malignancies. Our results demonstrate that this hairless mouse model for UV-induced skin cancer corresponds closely to human non-melanoma skin cancers with respect to mutations in the p53 gene.

Cells with UV-specific DNA damage are present in murine lymph nodes after in vivo UV irradiation.

Ultraviolet radiation is absorbed in the skin, especially in the epidermis. After ultraviolet irradiation the number of major histocompatibility complex class II+, adenosine triphosphatase+ Langerhans cells and Thy-1+ dendritic epidermal cells in the epidermis decreases. Whether this decrease is due to migration of these cells or to loss of membrane markers is not clear. To address this question we have used the monoclonal antibody H3 directed against cyclobutyl thymine dimers-a form of DNA damage that is specifically induced by ultraviolet radiation-to investigate whether H3+ cells are present in the draining lymph nodes of the skin after ultraviolet irradiation of hairless, inbred mice (HRA/Skh). After a single dose of ultraviolet radiation (Westinghouse FS40, 1.5 kJ/m2), H3+ cells were present in the paracortex of the draining lymph nodes. No positive cells were found in the blood of irradiated mice. These results suggest that the H3+ cells in the lymph nodes originate from the skin. The number of H3+ cells in the draining lymph nodes increased the first 24 h after irradiation and then stabilized. Immunohistochemical double staining revealed that all H3+ cells were major histocompatibility complex II+, and that only a fraction of the cells were NLDC-145 positive. No V gamma 3 T-cell receptor bearing cells could be found in the lymph nodes after UV irradiation of the skin.

Ultraviolet radiation--induced impairment of the early initiating and the late effector phases of contact hypersensitivity to picrylchloride: regulation by different mechanisms.

Two types of antigen-specific T cells are needed for the elicitation of contact hypersensitivity reactions. They act in an obligate sequence to mediate the early initiating and late effector phases of contact hypersensitivity, which are accompanied by skin-swelling responses at 2 and 24 h after challenge, respectively. The magnitude of the late ear swelling depends on that of the early swelling. We studied the influence of ultraviolet radiation on both phases of contact hypersensitivity to picrylchloride. Mice were exposed to subedemal doses of ultraviolet radiation on the shaved backs for four consecutive days. Four days later mice were sensitized on non-irradiated skin. Four days after sensitization mice were challenged on the ears, and swelling was measured 2, 4, and 24 h after challenge. The early and late phases of contact hypersensitivity were largely suppressed in ultraviolet-irradiated, actively sensitized mice. Transfer of immune lymphoid cells from donor mice that were sensitized 4 d earlier induced early and late components of contact hypersensitivity in naive recipients after challenge. Transfer of immune lymphoid cells from donors that were sensitized 1 d earlier only induced the early component of contact hypersensitivity. Ultraviolet irradiation of donor mice significantly reduced the capacity of the immune lymphoid cells to induce contact hypersensitivity. We show that lymphoid cells responsible for the early and late components of contact hypersensitivity are both affected.

Immunochemical detection of cyclobutane thymine dimers in epidermal Langerhans cells of ultraviolet B-irradiated hairless mice.

An immunocytochemical method was developed to study in vivo induction and removal of DNA damage in a specific cell population in the epidermis of hairless mice after ultraviolet B (UVB) exposure: the immunocompetent antigen-presenting Langerhans cells. To this aim, Ia+ cells, which are representative for epidermal Langerhans cells, were compared with the bulk of epidermal cells with respect to the nuclear level of cyclobutane thymine dimers. Mouse Langerhans cells were identified with a membrane-located immunoperoxidase stain, whereas DNA and DNA damage were revealed with fluorescent nuclear stains. After a low dose of UVB (approximately 1 minimal erythema dose), dimer levels were determined both in all murine epidermal cells and in Ia+ cells separately. At 24 h after irradiation, dimer removal was still incomplete, with a persistence of approximately 50% of the initially induced dimers in epidermal cells in general, and of approximately 75% in Langerhans cells. Possible applications of the method developed and the results presented here are discussed in relation to the immunosuppressive effect of UV.

Lymphocyte migration into the lamina propria of the gut is mediated by specialized HEV-like blood vessels.

Migration of lymphocytes into the lamina propria of the small intestines was studied in mice using short-term in vivo migration experiments in combination with immunocytochemistry and autoradiography. The results show that, shortly after intravenous injection, most of the lymphocytes present in the lamina propria are actually located within the capillary network of the villi. Furthermore, it was shown that lymphocytes leave the blood stream and enter the lamina propria via small blood vessels at the base of the villi. These blood vessels can be discriminated by their positive staining with MECA-325, a monoclonal antibody that is specific for high endothelial venules (HEV) in lymphoid organs. From the results it is concluded that the gut contains specialized venules at specific sites, involved in the emigration of lymphocytes, comparable to HEV in lymphoid organs. The flatness of the endothelium of these MECA-325-positive intestinal blood vessels, which is in contrast to the situation in lymphoid organs, could not be changed by inducing an intestinal inflammation. This flatness may be directly correlated to the less efficient transmigration of lymphocytes, as demonstrated in our experiments.