Frances P. Noonan

Neonatal sunburn and melanoma in mice.

Retrospective epidemiological data have indicated that cutaneous malignant melanoma may arise as a consequence of intense, intermittent exposure of the skin to ultraviolet radiation, particularly in children, rather than from the cumulative lifetime exposure that is associated with other forms of skin cancer. Here we use a genetically engineered mouse model to show that a single dose of burning ultraviolet radiation to neonates, but not adults, is necessary and sufficient to induce tumours with high penetrance which are reminiscent of human melanoma. Our results provide experimental support for epidemiological evidence that childhood sunburn poses a significant risk of developing this potentially fatal disease.

Immunosuppression by ultraviolet B radiation: initiation by urocanic acid

Irradiation with UV-B, a component of natural sunlight, initiates systemic immunosuppression of delayed-type hypersensitivity responses. This may be a fundamental regulatory mechanism, controlling the interaction between mammals and potentially deleterious environmental UV radiation. Here, Frances Noonan and Edward De Fabo assess the evidence that suppression is initiated by the photoisomerization of trans-urocanic acid (UCA) in the stratum corneum, discuss the significance of this mechanism for skin cancer outgrowth and propose applications for UCA in transplantation.

Melanoma induction by ultraviolet A but not ultraviolet B radiation requires melanin pigment.

Malignant melanoma of the skin (CMM) is associated with ultraviolet radiation exposure, but the mechanisms and even the wavelengths responsible are unclear. Here we use a mammalian model to investigate melanoma formed in response to precise spectrally defined ultraviolet wavelengths and biologically relevant doses. We show that melanoma induction by ultraviolet A (320–400 nm) requires the presence of melanin pigment and is associated with oxidative DNA damage within melanocytes. In contrast, ultraviolet B radiation (280–320 nm) initiates melanoma in a pigment-independent manner associated with direct ultraviolet B DNA damage. Thus, we identified two ultraviolet wavelength-dependent pathways for the induction of CMM and describe an unexpected and significant role for melanin within the melanocyte in melanomagenesis.

Early pregnancy factor is immunosuppressive

IMMUNOSUPPRESSION during pregnancy has important consequences not only with respect to the recognition of the antigenically dissimilar embryo1 but also in terms of susceptibility to viral or tumour assaults2,3. We have recently described an early pregnancy factor (EPF) detectable within 24 h of mating and have postulated that this protein is responsible for depression of lymphocyte activity during pregnancy4–6, Immunosuppressive properties have been attributed to other proteins detectable during pregnancy. These include pregnancy-associated macroglobulin7,8 α-fetoprotein9 and human chorionic gonadotropin (HCG)10,11. One characteristic these substances have in common, but in which they differ from EPF, is that they are not present, or not detectable, in the early stages of pregnancy. Recently the immunosuppressive activity of HCG has been disputed by several workers working with highly purified material12,13. We have now shown that EPF inhibits the expression of an immune response in vivo.

Susceptibility to immunosuppression by ultraviolet B radiation in the mouse

Irradiation with ultraviolet B (UVB; 290–320 nm) initiates systemic immunosuppression of contact hypersensitivity (CHS). UV dose-responses for suppression of CHS to trinitrochlorobenzene were established in 18 strains of inbred mice. Three phenotypes with significantly different susceptibilities to UV suppression were identified. The phenotypes were: high (HI) susceptibility, 50% suppression with 0.7–2.3 kJ/m2 UV (C57BL/6, C57BL/10, and C57L and NZB females); low (LO) susceptibility, 50% suppression with 9.6–12.3 kJ/m2 UV (BALB/c, AKR, SJL and NZW), and intermediate (INT) susceptibility, 50% suppression with 4.7–6.9 kJ/m2 UV (DBA/2, C57BR, C3H/HeJ, C3H/HeN, CBA/N and A/J). UV suppression was not correlated with skin pigmentation or with the magnitude of the CHS response in non-irradiated animals. Major histocompatibility complex (MHC) haplotype was not correlated with UV suppression in MHC congenic strains B10.D2/oSnJ, B10.D2/nSnJ, B10.BR/SgSnJ, and A.BY/SnJ. There were no sex differences in UV suppression in BALB/c, C57BL/6, or NZW animals. In the autoimmune NZB strain, however, male mice (LO) were seven times less sensitive to UV suppression than NZB female mice (HI). Both sexes of (NZB × NZW)F1 and (NZW × NZB)F1 mice were HI, supporting dominance of HI over LO. Thus there are genetic factors and interacting sex-limited factors determining susceptibility to UV suppression. These findings may be of relevance to UV-related diseases such as photosensitive lupus and skin cancer.

BIOLOGICALLY EFFECTIVE DOSES OF SUNLIGHT FOR IMMUNE SUPPRESSION AT VARIOUS LATITUDES AND THEIR RELATIONSHIP TO CHANGES IN STRATOSPHERIC OZONE

Abstract— Using information on solar irradiance at different latitudes derived from a radiative transfer model and a detailed in vivo action spectrum for immune suppression in a murine system, we report here calculations of the “biologically effective” irradiance of sunlight for immune suppression. From 40°N to 40°S in summer, under normal stratospheric ozone concentrations this value ranged from 0.27 W/m2 (40°N or S) to a peak of 0.33 W/m2 (20°N or S) predicting that 50% immune suppression in the Balb/c mouse would occur after 21–26 min of sunlight exposure within this latitude range. We also found that the most effective wavelengths for immune suppression shift from a peak of 270 nm in the laboratory to near 315 nm in sunlight. Furthermore, using ozone depletion scenarios of 5 to 20%, at latitudes 20°S and 40°N, a 0.6% increase in biologically effective irradiance levels of solar UVB for immune suppression was predicted for each 1% decrease of ozone. This value rose to a nearly 1% increase for each 1% decrease in ozone at 60°N latitude in wintertime. These data indicate that activation of immune suppression, in a murine model, requires relatively low levels of sunlight and that these levels are easily obtainable over most of the populated regions of the world. Since a UVB‐activated photoreceptor, urocanic acid, regulates immune suppression in mice and since this same compound exists on other mammalian skin, including human skin, suppression of the mammalian immune system is predicted to increase if substantial stratospheric ozone depletion takes place.

Cis‐UROCANIC ACID DOWN‐REGULATES THE INDUCTION OF ADENOSINE 3', 5'‐CYCLIC MONOPHOSPHATE BY EITHER trans‐UROCANIC ACID OR HISTAMINE IN HUMAN DERMAL FIBROBLASTS in vitro

Abstract— It has been demonstrated that UVB radiation (290–320 nm) suppresses mammalian cell‐mediated immunity by effecting the trans to cis isomerization of urocanic acid (UCA) in the stratum corneum, the uppermost layer of the skin. Trans‐urocanic acid has been shown to be the photoreceptor for UVB‐induced immune suppression and the cis‐isomer has been demonstrated to be immunosuppressive. Little is known, however, about how the isomerization of UCA may affect the proximal or distal cells of the skin or the immune system. We report here that trans‐UCA is biologically active in vitro in human dermal fibroblasts, inducing adenyl cyclase as measured by cAMP (adenosine 3, 5‐cyclic monophosphate) formation in a dose‐dependent manner similar to the action of histamine. Trans‐UCA and histamine stimulate 50% of maximum activity at concentrations of 3.3 μM end 13.8 μM respectively. Cis‐UCA does not increase cAMP in these human fibroblasts but actively down regulates the increase of cAMP induced by either histamine or trans‐UCA. Cis‐UCA down regulated the histamine response by 75% and the trans‐UCA response by 60% at a concentration range of 1 mM to 1 nM. The trans‐UCA induction of cAMP can also be downregulated with an H2 histamine receptor antagonist cimetidine. These results support the hypothesis that a cellular target for cis‐UCA is the dermal fibroblast and the effects reported here may represent the initial biochemical and cellular event for UVB‐induced immune suppression i.e. the immediate step following the isomerization of trans to cis‐UCA is the down regulation of cAMP by cis‐UCA. Regulation of such an important second messenger such as cAMP could then allow cascading signals to occur, leading to immune suppression.

Control of UVB immunosuppression in the mouse by autosomal and sex-linked genes

Irradiation with UVB (290–320 nm) initiates a systemic immunosuppression detectable as suppression of contact hypersensitivity (CHS). We investigated susceptibility to UV suppression in reciprocal F1-hybrid and backcross mice derived from BALB/c (low susceptibility) and C57BL/6 (high susceptibility) inbred strains. CB6F1 male mice exhibited high susceptibility and B6CF1 male mice exhibited low susceptibility, indicating a major X-linked effect in the genetic control of UV immune suppression. Females of either F1 hybrid showed intermediate suppression, consistent with random X-inactivation. A model of monogenic X-linked control was not sufficient, and evidence for the action of two genetically unlinked autosomal genes was found in parental backcross animals. Both sexes of (BALB/c × CB6F1) mice showed a 1 high: 1 low ratio of phenotypes, indicating control by a major autosomal locus, Uvs1, confirmed by propagation of the high phenotype through selective backcrossing for nine generations to BALB/c. Uvs1 was not genetically linked to 12 chromosomal markers including the pigment genes b (brown) and c (albino). Backcross animals (C57BL/6 × CB6F1) showed a significant sex difference, male mice giving a 3 high: 1 low ratio of phenotypes, compatible with the action of a second autosomal locus, Uvs2, in this hybrid. The findings are compatible with a model in which high phenotype (Uvs1b/Uvs1b) is dominant when subjected to recessive epistatis by the X-chromosome locus Uvs3, or by the autosomal locus Uvs2. The finding of genetic control by interacting autosomal and X-linked genes is unique. Genetically determined high susceptibility to UV immunosuppression may be an important risk factor for UV-related human diseases.

Dietary β-carotene and ultraviolet-induced immunosuppression

Ultraviolet (UV)‐induced immunosuppression is a critical step in UV carcinogenesis, permitting tumour outgrowth. We investigated the effect of dietary β‐carotene on UV suppression of contact hypersensitivity (CHS) to trinitrochlorobenzene (TNCB) in BALB/c mice. Mice were fed for 10–16 weeks chow alone or supplemented with 1% β‐carotene or placebo as beadlets. Serum β‐carotene was detectable by high performance liquid chromatography (HPLC) analysis only in β‐carotene‐fed mice (2.06u2003±u20030.15u2003μg/ml). Serum retinol was 0.22–0.27u2003μg/ml in all three groups. Mice (nu2003=u200341/dietary group) were irradiated with 0, 4.5, 9 or 18u2003kJ/m2 of UVB and the CHS response was measured. Decreased CHS responses were observed in all UV‐irradiated groups compared with unirradiated controls. UV dose–responses for suppression of CHS derived by first‐order regression analyses of plots of percentage suppression of CHS as a function of log10UV dose showed significant slopes (Pu2003<u20030.02) for all three dietary groups and similar residual variances between groups, Pu2003>u20030.05. The UV dose for 50% suppression of CHS was 6.3u2003kJ/m2 for control, 6.4u2003kJ/m2 for placebo, and 5.5u2003kJ/m2 for β‐carotene‐fed mice. No significant differences in slopes or elevations between UV dose–responses were observed, Pu2003>u20030.05. Skin levels of the initiator of UV‐induced immunosuppression, cis urocanic acid, were determined by HPLC in mice given 0 or 9u2003kJ/m2 of UV (nu2003=u200328/dietary group). No significant differences were observed between dietary groups (range 35.2–41.1u2003ng/mg skin, Pu2003>u20030.15) We conclude feeding β‐carotene to BALB/c mice does not alter susceptibility to UV immune suppression, in contrast to human studies.

A melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma

Melanocortin 1 receptor (MC1R) signaling stimulates black eumelanin production through a cAMP‐dependent pathway. MC1R polymorphisms can impair this process, resulting in a predominance of red phaeomelanin. The red hair, fair skin and UV sensitive phenotype is a well‐described melanoma risk factor. MC1R polymorphisms also confer melanoma risk independent of pigment. We investigated the effect of Mc1r deficiency in a mouse model of UV‐induced melanoma. C57BL/6‐Mc1r+/+‐HGF transgenic mice have a characteristic hyperpigmented black phenotype with extra‐follicular dermal melanocytes located at the dermal/epidermal junction. UVB induces melanoma, independent of melanin pigmentation, but UVA‐induced and spontaneous melanomas are dependent on black eumelanin. We crossed these mice with yellow C57BL/6‐Mc1re/e animals which have a non‐functional Mc1r and produce predominantly yellow phaeomelanin. Yellow C57BL/6‐Mc1re/e‐HGF mice produced no melanoma in response to UVR or spontaneously even though the HGF transgene and its receptor Met were expressed. Total melanin was less than in C57BL/6‐Mc1r+/+‐HGF mice, hyperpigmentation was not observed and there were few extra‐follicular melanocytes. Thus, functional Mc1r was required for expression of the transgenic HGF phenotype. Heterozygous C57BL/6‐Mc1re/+‐HGF mice were black and hyperpigmented and, although extra‐follicular melanocytes and skin melanin content were similar to C57BL/6‐Mc1r+/+‐HGF animals, they developed UV‐induced and spontaneous melanomas with significantly less efficiency by all criteria. Thus, heterozygosity for Mc1r was sufficient to restore the transgenic HGF phenotype but insufficient to fully restore melanoma. We conclude that a previously unsuspected melanin‐independent interaction between Mc1r and Met signaling pathways is required for HGF‐dependent melanoma and postulate that this pathway is involved in human melanoma.