IMMUNOSUPPRESSIVE, CARCINOGENIC AND METASTASE -RELATED EFFECTS OF SOLAR UV RADIATION

Project leader: Christer Jansén, University of Turku, Department of Dermatology, Kiinamyllynkatu 4-8, FIN-20520 Turku, Finland, tel: +358-2-313 2605, fax: +358-2-313 1610, e-mail: Cjansen@utu.fi
 
 

PUBLICATIONS

TIIVISTELMÄ SUOMEKSI

Researchers:
Dariusz Leszczynski, Radiobiology Laboratory, Department of Research and Environmental Surveillance, STUK - Radiation and Nuclear Safety Authority, Laippatie 4, FIN-00880 Helsinki, Finland, tel. +358-9-7598 8694, fax: +358-9-7598 8464, e-mail: Dariusz.Leszczynski@stuk.fi
Riikka Pastila, Radiobiology Laboratory, Department of Research and Environmental Surveillance, STUK - Radiation and Nuclear Safety Authority , Laippatie 4, FIN-00880 Helsinki, Finland, tel. +358-9-7598 8468, fax: +358-9-7598 8464, e-mail: Riikka.Pastila@stuk.fi
Leena Koulu, Department of Dermatology, University of Turku, Kiinamyllynkatu 4-8, FIN-20520 Turku, Finland, tel. +358-2-313 2600, fax: +358-2-313 1610
e-mail: Leena.Koulu@tyks.fi
Lasse Leino, BioTie Therapies, Turku Technology Center, Biocity, Tykistökatu 6 20520 Turku, Finland, tel. +358-2-274 8914, fax: +358-2-274 8910,
e-mail:  Lasse.Leino@biotie.fi
Jarmo Laihia, Department of Dermatology, University of Turku, Kiinamyllynkatu 4-8, 20520 Turku, Finland, tel. +358-2-261 1634, fax: +358-2-313 1610, e-mail: Jarlai@utu.fi

Financing SYTTY organisation:  The Academy of Finland
Funding from SYTTY / Total funding of project (€): 213274 / 671590
Person-months of work funded by SYTTY / Total person-months of work: 78,5 / 105,6

KEY  WORDS: Solar UV, immunosuppression, skin cancer, metastasis, risk assessment
 

EXTENDED ABSTRACT

1 Introduction

Epidemiological studies have demonstrated that ultraviolet (UV) radiation induces skin cancers. It has also been realised that UV radiation affects the immune system in ways that are instrumental not only in the development of skin cancer but also in local and systemic infections. Also, it has been demonstrated that UVA radiation may have much more profound physiological effects, both local (skin) and systemic, than previously anticipated. Our project has been launched to provide information of UV-compromised human immune surveillance of skin cancer, in particular malignant melanoma, and to examine the possible effect of UV radiation on tumour metastasis.

UV immune modulation was studied in humans in vivo. We used contact hypersensitivity (CHS) induction to an experimental allergen as a model for cutaneous cell-mediated immunity. In comparison, we studied the molecular mechanisms in human UV immune suppression by an epidermal cell-mediated T cell stimulation assay.

The majority of the biomedical studies on the development of skin cancer have focussed on the effects of UVB radiation (280–320 nm). However, several recent studies have demonstrated that UVA radiation (320–400nm) can modulate biochemical processes in the epidermis and dermis. Thus, we have suggested (Leszczynski D. et al., Photochem Photobiol 1996; 64: 936-942) the possibility that UVA radiation, either alone or in combination with UVB, might alter the metastatic potential of tumour cells passing through dermal capillaries or originating from the skin, by increasing their adhesiveness to the endothelium. The pro-metastatic effects of UVA might not be of significant relevance for the every-day exposures which are low, but UVA may gain prominence during sunbathing or tanning in solaria. Therefore, in this project we have aimed at the elucidation of the possible role of UVA irradiation in the enhancement of tumour cell–endothelial cell interaction. This enhancement of adhesiveness might lead to an increase in binding of UVA-irradiated tumour cells to endothelial lining of vasculature in various internal organs.

2 Methods

2.1. Immunosuppressive effects of solar UV radiation in humans
Healthy volunteers were sensitised with a single topical exposure to diphenyl cyclopropenone (DPCP) to induce long-lasting cell-mediated immune reactivity. Sensitisation was clinically tested at a distant skin site. Blood samples were drawn at fixed time points before and after sensitisation, T cells were enriched from peripheral blood lymphocytes (PBL), and cell proliferation was tested as a response to DPCP in vitro. The modulation of epidermal Langerhans cell (LC) function by solar-simulating UV irradiation (SUV; spectral range 290–400 nm) was examined by irradiating the buttock skin with a single dose (4 SED; 400 J/m2). Keratome or suction blister samples taken at fixed time points were dissected into epidermal cell (EC) suspension. Allogeneic PBL or autologous T cells were incubated with the EC and recall antigen(s), and the proliferative index was determined by 3H-thymidine incorporation. Also cutaneous malignant melanoma (CMM) patients were examined in the CHS induction test, but no blood or skin samples were taken.

2.2. Effects of UVA radiation on melanoma metastasis
In vitro experiments:
- Cell lines: Mouse B16-F1 and B16-F10 melanoma cell lines, mouse MS-1 endothelial cell line, human Mel-Juso melanoma cell line and human EAhy.926 endothelial cell line.
- Adhesion assay between UVA irradiated melanoma cell line and non-irradiated endothelial cell line was performed using method of Pauli and Lee (Lab Invest 1988; 58: 379).
- Expression of cadherin E, N and P on mouse melanoma cell lines and CD146 antigen on human melanoma cell line were determined by standard flow cytometry (FACS) and ELISA methods.
- To assess the homotypic interaction between melanoma cells, aggregation assay was performed where melanoma cells were allowed to form spontaneous aggregates in culture.
In vivo experiments:
- To validate our in vitro results, the study of experimental lung metastasis was performed using method by Fidler (Cancer Res 1973; 35: 218). Briefly, 50 000 melanoma cells were injected into the lateral tail vein in C57BL/6 mice and mice were exposed to 8 J/cm2 of UVA. Fourteen days later the mice were killed and the number of surface tumour nodules in lungs was counted.
- The adhesive properties of B16-F1 cells in vivo were examined at Wellman Laboratories of Photomedicine (Harvard University, Boston, MA, USA). Melanoma cells, irradiated with a single dose of UVA at 8 J/cm2, were injected into the tail vein 24 h after irradiation. Behaviour of injected melanoma cells in skin capillary circulation of the ear was monitored and recorded in the living animal using real-time video-rate fluorescence confocal microscope that was constructed and developed at Wellman Laboratories.

3 Results and Discussion

3.1 Immunosuppressive effects of solar UV radiation in humans
We have earlier observed that epidermal LC up-regulate crucial immune receptors upon SUV irradiation of the human skin in vivo. To assess the functional characteristics of LC in the irradiated skin, we used the mixed epidermal cell-lymphocyte reaction assay. PBL proliferation was suppressed by 48 % –61 % by allogeneic EC sampled at 3–48 h after SUV in vivo. SUV induced the expression of CD86 on LC at all time points. Accordingly, expression of CD25 and CD3 on the responding T cells appeared at the same or elevated levels. The data show that LC from the human skin exposed to SUV in vivo can suppress the proliferation response of allogeneic lymphocytes although the surface receptor expression on the responding T cells is not suppressed. In similar assays, however, SUV irradiation did not diminish the capacity of LC to present HSV and PPD recall antigens to enriched autologous T cells. CD25 and CD69 were induced on T cells in the antigen-presentation reaction. The two response types thus seem to function via distinct signalling pathways in local UV immunomodulation.

In our studies on local and distant UV immune modulation, all control subjects could be sensitised to DPCP, when the allergen was applied on nonirradiated skin, and a primary allergic reaction (PAR) developed. Local SUV (600 J/m2) prevented contact sensitisation through the irradiated skin in 11 out of 12 subjects, but it did not induce antigen-specific tolerance; the subjects could be sensitised later through the nonirradiated skin. A single UVB dose given to app. 70 % of body surface area did not prevent contact sensitisation through a distant, nonirradiated skin site (n=10). However, four consecutive minimal perceptible erythemal UVB doses prevented contact sensitisation at a distant skin site in two out of 21 subjects (no PAR at sensitisation site and no elicitation response later). One of these two subjects could not be sensitised to DPCP even later through the nonirradiated skin. This suggests that immunologic tolerance at a distant skin site can be achieved in humans, a phenomenon described earlier in experimental animals only. The basal ability of CMM patients to mount contact allergy to DPCP tended to be diminished as compared to individuals with no history of skin malignancy. The distant immunosuppression found in a small subpopulation of healthy humans could not be demonstrated in CMM patients.

When PBL were challenged with DPCP in vitro, they proliferated, up-regulated the CD25, CD69, and CD86 markers, and showed decreased expression per cell of CD4, CD8, and CD28 antigens. The latter effect was DPCP concentration-dependent. DPCP in 77 µM concentration was found to be the threshold level that blocked proliferation and IL-6 production, and induced apoptosis (annexin-V and propidium iodide with FACS). DPCP reactivity was demonstrable in PBL proliferation and in expression of cell surface components. DPCP seemed to affect signal transduction in T cells via antigen presentation and not via chemical toxicity. We compared the clinical in-vivo sensitisation to in-vitro proliferation and realised that, three weeks after sensitisation, DPCP-specific proliferation was detected only in some of the newly sensitised subjects who responded positively in the clinical skin testing. This showed that cell proliferation was not an adequate parameter to indicate systemic sensitisation to the experimental haptenic allergen. The ELISPOT analysis will be validated as an alternative method.

3.2 Effects of UVA radiation on melanoma metastasis
- In vitro study with mouse cell lines (manuscript submitted for publication)
We have determined in vitro that UVA irradiation of mouse B16-F1 and B16-F10 melanoma cells causes increase in melanoma cell adhesiveness to non-irradiated mouse endothelial monolayers. The single dose of UVA at irradiance of 8–12 J/cm2 caused statistically significant increase in melanoma adhesiveness peaking at 24 h after irradiation in both cell lines. The same dose of UVA radiation, but delivered as four smaller doses separated by 1-h time-intervals (4 x 2 J/cm2), induced increased B16-F1 melanoma cells adhesion already at 1-h time-point. This suggests a possible cumulative effect of multiple doses of UVA irradiation. UVA irradiation induced decline in the surface expression of E-cadherin and increase in the expression of N-cadherin in B16-F1cells. This change is a well-known marker of metastatic melanoma phenotype. The decline in E-cadherin expression was accompanied by a significant decline in homotypic melanoma-melanoma adhesion (clustering) that is regulated by E-cadherin. This suggests that, following UVA irradiation, the strength of adhesion between melanoma cells in the primary tumour might weaken, which might facilitate detachment and migration of single cells from the solid tumour mass into capillary circulation.
- In vitro study with human cell lines
Results of the experiments executed using mouse cells are being repeated with human cell lines. Part of the adhesion and clustering data has been already obtained. Experiments will be continued and finished in 2002 (internal STUK funding).
- In vivo study in mice
The physiological significance of the results obtained in vitro is being confirmed by executing an animal study in vivo. The aim of this study is to determine whether melanoma cells, i.v. injected into C57BL/6 mice that were subsequently either non-irradiated or irradiated with 8 J/cm2 or 3 x 8 J/cm2 dose of UVA, formed lung metastases. Total of 160 animals in 16 groups were used in the study. Two weeks after injection of melanoma cells animals were sacrificed, and lung, liver, brain and skin samples were collected to determine the quantity and quality of metastases (lung, liver, brain) and the effect of UVA radiation (skin). Study will be finished in 2002 as a part of the Ympäristöterveyden Tutkijakoulu (SYTYKE).
- In vivo confocal microscopy study in mice
Using in vivo confocal microscope it was possible to observe sticking, rolling and extravasation of the i.v. injected rhodamine-labelled B16-F1 melanoma cells. However, most likely, illumination during microscopic observation caused a rhodamine-dependent photodynamic reaction in melanoma cells, which led to enhanced cell extravasation, independently of whether cells were or were not UVA-treated. Following several attempts to resolve this problem by altering the labelling method and the amount of illuminating light, experiments were terminated.

4 Conclusions

- Distant cutaneous immunosuppression to a synthetic allergen can be induced by SUV in human subjects
- The basal ability of CMM patients to mount contact allergy to DPCP seems to be diminished as compared to individuals with no history of skin malignancy
- Local SUV induces B7-2 costimulatory molecule in epidermal Langerhans cells in vivo, but reduces the capacity of these cells to stimulate allogeneic lymphocyte proliferation in vitro
- Local SUV in vivo does not affect the capacity of Langerhans cells to present recall protein antigens to autologous T cells in vitro
- Systemic sensitisation to a synthetic allergen can be indicated by lymphocyte proliferation and cell surface antigen expression in vitro in certain unknown conditions only
- UVA irradiation increases the following pro-metastatic properties of melanoma cells:
    - UVA irradiation enhances adhesiveness of melanoma cells to endothelium
    - UVA irradiation down-regulates cadherin E-dependent homotypic melanoma clustering
    - UVA irradiation up-regulates cadherin N expression, which may facilitate transition of melanoma cells to capillaries
 

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