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ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

The neonatal immune environment and the events that occur during this time have profound effects for the adult period. While protective immune responses can develop, the neonatal immune system, particularly the skin immune system (SIS), tends to promote tolerance. With this information we undertook a number of studies to identify unique aspects of skin during the neonatal period. Proteomics revealed proteins uniquely expressed in neonatal, but not adult, skin (e.g. Stefin A, peroxiredoxins) and these may have implications in the development of SIS. Vitamin D was found to have a modulating role on SIS and this was apparent from the early neonatal period. Exposure of the neonatal skin to UV radiation altered the microenvironment resulting in the generation of regulatory T cells, which persisted in adult life. As the development of UV radiation-induced melanoma can occur following a single high dose (equivalent to burning in adults) to transgenic mice (hepatocyte growth factor/scatter factor or TPras) during the neonatal period, the early modulating events which lead to suppression may be relevant for the development of UV radiation-induced human melanoma. Any attempt to produce effective melanoma immunotherapy has to accommodate and overcome these barriers. Margaret Kripke’s pioneering work on UV-induced immunosuppression still remains central to the understanding of the development of melanoma and how it frequently escapes the immune system.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

The incidence of melanoma continues to rise and in the past 20 years the number of cases has doubled (1). While the role of UV radiation in the causation and pathogenesis of cutaneous melanoma was once a matter of controversy (2,3), epidemiologic studies from Australia have provided links between sun exposure in childhood and melanoma (4–6). Whiteman et al. (7) in a detailed systematic review of epidemiologic studies concluded that high level of exposure to sunlight in childhood is a strong determinant of melanoma risk, but adult exposure may also play a role. Whiteman and Green (8) had previously highlighted the importance of sunburn associated with severe inflammation as a risk factor in melanoma development. Weinstock et al. (9) supported this concept as they showed that blistering sunburn between 15 and 20 years of age was associated with an increased risk of melanoma, while no association was found with blistering sunburn after the age of 30 years.

The physical barrier provided by the skin is critical to protect against external agents that may cause damage to the host. However, the skin is more than just a physical barrier and the concept of the skin immune system (SIS) proposed by Bos and Kapsenberg is now firmly established (10,11). The cells associated with the development of cutaneous immune reactions include dendritic antigen presenting cells such as Langerhans cells (LC) as well as monocytes/macrophages, mast cells, lymphatic/vascular endothelial cells and T lymphocytes. Important humoral components include cytokines, neuropeptides, prostaglandins and free radicals. The SIS can respond to local environmental agents by either producing an active immune response to eliminate the agent or by inducing tolerance to avoid the possibility of causing damage to the host by an overactive immune response. The direction of the response is very much dependent on the status of the microenvironment.

UV radiation is one important factor, which not only alters the skin environment, but can also induce skin cancer. Margaret Kripke realized that although these tumors were highly antigenic, they escaped immunologic destruction and her pioneering studies were designed to resolve the question as to how these immunogenic tumors avoid immune recognition. Kripke’s work demonstrated that during the course of exposure to chronic UV radiation, mice lost their capacity to reject transplanted highly antigenic UV-induced skin tumors. This change was present long before there was any evidence of development of skin cancer. These experiences led Kripke and her postdoctoral fellow, Michael Fisher, to propose the concept of UV radiation-induced systemic immune suppression (3,12); the immunologic nature of these events was later confirmed by transferring suppression with lymphoid cells. Later, suppressor T lymphocytes, specific for UV-induced tumors were found in the lymphoid organs of UV-irradiated mice (13–15). Kripke and Fisher then showed the functional significance of these cells by controlling the development of primary skin cancer in UV-irradiated mice (16). These tolerogenic events were further explored by Kripke and co-workers by applying contact sensitizing antigens such as 2,4,6-trinitrochlorobenzene (TNCB), dinitrofluorobenzene (DNFB) or fluorescein isothiocyanate (FITC) to the UV-treated epidermis (17–19). Chemical carcinogens and tumor promoters such as dimethylbenz (a) anthracene (DMBA) and 12-0-tetradecanoylphorbol-13-acetate, respectively, applied to mouse skin, likewise alter the cutaneous environment so that the application of antigen results in tolerance and the generation of antigen-specific suppressor T cells (20,21). These studies clearly show that alteration to the skin environment by either UV light or chemical carcinogens sets in motion cellular events resulting in suppressor T cell generation which allow antigenic tumors to develop (22).

As noted above, exposure to sunlight in childhood, especially to burning doses, is a risk factor for the development of melanoma (7). This occurs at a time when the skin can be considered immature and is undergoing developmental changes. Little is known about SIS during this developmental period in humans, whereas in the mouse model it has been established that neonatal skin is associated with a poverty of immune and inflammatory responses (23,24) and antigen applied at this time tends to result in tolerance. This article reviews our own studies on neonatal SIS, the modulating influence of vitamin D and the effects of exposure to antigen and UV light at this time. With this background information in murine models, the challenges of understanding the genesis of melanoma can be explored.

Neonatal Skin Structure

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

Compared with adult skin the structure of neonatal murine skin is highly cellular. The epidermis is approximately four to five cells thick, with a prominent keratin layer which forms a veritable barrier to both outside insult and fluid loss from internal structures. The neonatal dermis contains many mesenchymal cells, embedded in a semifluid ground substance. Early hair follicles arise throughout the lower dermis, which comprises a thick zone of developing adipose tissue. Other cells include prominent mast cells, often in relation to the small blood vessels. Over a period of 3 weeks, the skin continues to develop through a process of proliferation, maturation and remodeling. The epidermis arises from a single layer of multipotent epithelial stem cells (25) and the dermis, consisting of many structural fibers gives rise to mature connective tissue. By maturity the remodeling process reduces the mouse epidermis to a thickness of two to three cells (Fig. 1).

image

Figure 1.  Comparison of neonatal (day 4) and adult (week 6) skin. Hematoxylin and eosin-stained sections reveal that neonatal skin has a thicker protective keratin layer, thicker epidermis and is generally more cellular than adult skin.

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With proteomics we have revealed widespread changes in protein expression in murine skin at birth relative to adulthood (26). By comparing neonatal skin with adult skin we identified approximately 179 proteins which were differentially expressed in neonatal and adult tissue. One such protein was Stefin A, which was highly expressed in the neonatal skin and decreased with age, suggesting a functional change during development. As this protein is also up-regulated in proliferative disease states of the skin, it is likely that this protein is involved in cellular proliferation and could provide a useful target for diseases of abnormal proliferative conditions, including psoriasis and cancer. Another family of proteins we have recently identified, by comparing the protein profiles in the epidermis, is the peroxiredoxins, which is expressed at greater levels in neonatal skin. This group of proteins is effective at scavenging reactive oxygen species, such as hydrogen peroxide, and can therefore protect the skin from damage by these agents (27,28). Peroxiredoxin-2 has recently been implicated in the inhibition of T lymphocytes and dendritic cells (DC) (29), suggesting an important regulatory role in the developing SIS.

Like the development of the SIS the development of the epidermis is governed by signaling pathways and growth factors. At the onset, the surface of the embryo forms into a single layer of multipotent epithelial cells, driven by Wnt signaling (30). This is an inhibitory signal which blocks the developing outer layer of progenitor cells from responding to fibroblast growth factors (FGFs), and in turn allows them to respond to bone morphogenic proteins (BMP) (31). BMP signaling designates the fate of these cells to form the single-layered epidermis. Under further signaling via epidermal growth factor and notch pathways, the single layer becomes stratified via the resulting differentiation and migration. At the morphologic level the interactions between signaling pathways and growth factors govern the differentiation of the basal cells to mature squamous keratinocytes characterized by changes in keratins, lipid markers and other proteins which ultimately signal programmed cell death.

Likewise, melanocytes derived from pluripotent neural crest cells use various signaling pathways for melanocyte development and migration (32). WNT/β-catenin signaling is essential for both neural crest induction and melanocyte development (33) while c-kit, steel factor (SCF) and the neural crest transcription factor, SLUG, are involved in melanoblast expansion, survival and migration that allows these cells to become established in hair follicles and epidermis (reviewed in Lin and Fisher [32]). Endothelins 1 and 3 and hepatocyte growth factor (HGF) and basic FGF seem to be involved in later stages of melanocyte migration from the dermis into the epidermis (34). As dermal melanoblasts move through the basement membrane they express E-cadherin which is then down-regulated and replaced by P-cadherin as the cells pass into the hair follicles (35).

Microphthalamia-associated transcription factor is activated early as the neural crest cells migrate to become melanoblasts and it is required for their survival (36). Other inhibitors of apoptosis such as bcl-2 support melanocyte viability (37). Melanocyte stem cells are present in the bulge region of hair follicles which migrate as differentiated melanocytes to the hair bulb where they export pigment to hair-producing keratinocytes (32).

Understanding melanoma stem cells and signaling pathways has proved important in exploring the basis of melanoma. Transgenic mouse models overexpressing ras (TPras) and HGF (HGF/scatter factor [SF] transgenic), both develop melanomas after a single neonatal dose of UV radiation (38,39).

A characteristic feature of neonatal skin, with regard to development, is the gradual structural and functional changes in LC, particularly with regard to their alterations in cell populations, distribution, morphology and cell marker expression. In neonatal skin there is a low LC density with these cells displaying a reduced number and shorter dendrites. The cells have a reduction in molecules critical for the immune response (40). These include MHC-II, costimulatory molecules (CD40, CD80, CD86) and reduced antigen uptake receptors (41,42). As adults, the LC density increases, with a concordant increase in the expression of these receptors resulting in the ability to induce active immune responses (40).

Response of Neonatal Skin to Antigen

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

The neonatal period is a unique time of life as it is a phase characterized by susceptibility to infections and to carcinogenesis (43,44). Although the mechanisms remain poorly defined, the immature status of the immune system has been considered a major factor. It is well established that following exposure to antigen, neonatal mice tend to develop Th2 responses and tolerance (44–47). Originally this was attributed to the immaturity and reduced number of T cells (44,47), but although this general simplification provided a plausible explanation for the tolerogenic nature of the neonatal immune response, in 1996 three key papers showed that this was inaccurate (48–50). These changed our perception of the neonatal immune system as it is now clear that neonatal mice can produce mature immune responses under certain conditions. Nonetheless, immune responses in early life differ from adult life due to a number of differences between the neonatal and adult immune systems (44). Critically important is the microenvironment, as well as the mode of delivery of antigen to T cells. The direction of immune responses that are established in early life can influence the outcome of immune responses in later adult life.

As DC are critically important to the induction, as well as the direction, of an immune response, attention has been directed toward these cells to explain differences in neonatal and adult immune responses. Our analyses of LC during development have indicated that these cells are phenotypically and functionally different to LC of adult mice. On the basis of MHC-II, DEC-205 and Langerin expression, the LC network of 3-day-old neonatal mice is poorly formed. The cells appear to have rounded bodies with limited detectable dendrites (40). During development these cells acquire their dendritic form and by 14 days of age the cells have their adult-like appearance (40). This development of the LC network directly correlates with the functional capacity of the LC to induce T cell proliferation. This was determined by applying the fluorescent contact sensitizer, FITC, through the skin and collecting the FITC-positive DC from the draining lymph nodes and using these cells to stimulate adult T cells (41). Such FITC tracking techniques have been successfully employed by Margaret Kripke in the past to explore cellular immune events during UV-induced tolerance induction and is a technique that was successfully utilized by Knight and colleagues to elucidate the role of DC in the initiation of immune responses (51–54).

Further investigations into the reduced ability of LC from murine neonatal skin to induce T cell proliferation provided valuable insights into neonatal tolerance. Again, using the FITC-tracking technique we found that LC from neonatal skin had a reduced ability to transport antigen to the draining lymph nodes as there were less cells reaching the lymph node and these cells transported a lower amount of antigen when compared with LC from adult skin (40). In addition, there was a diminished expression of the molecules involved with antigen presentation and costimulation, specifically MHC-I, MHC-II plus CD40, CD80 and CD86, with the reduced expression of CD40 translating to a loss in cell signaling (41). Consequently the combined effect of reduced antigen carriage and reduced accessory molecule expression culminates in less signaling to antigen-specific T cells, a failure to induce full T cell activation and tolerance induction.

The neonatal epidermis shares some similarities with the epidermis of UV-treated and carcinogen-treated skin. Both UVB, and the chemical carcinogen DMBA, cause migration of LC away from the epidermis, leaving the skin susceptible to antigen-specific tolerance induction. When antigen is applied to carcinogen-treated skin, the residual LC that migrate to the draining lymph nodes have reduced antigen uptake (55), similar to the response observed in neonatal skin. Failure to acquire antigen would appear central to the inability of LC from neonatal epidermis to induce effective immunity. LC from the epidermis of 3-day-old neonatal mice do not express DEC205 and have a reduced expression of Langerin (40,42,56), suggesting an impaired ability to endocytose antigen. On analyzing antigen uptake by neonatal LC we discovered that they were relatively more efficient than adult LC but, rather than utilizing an endocytic pathway, as performed by adult LC, neonatal LC preferentially utilized a fluid phase pathway (42). Because the mode of antigen uptake influences the outcome of the internalized antigen (57) we monitored the fate of antigen internalized by neonatal LC. Despite antigen internalized via a fluid uptake mode, neonatal LC could still proteolyze antigen and re-express this antigen on the cell surface in association with MHC-II (B. M. Bellette, D. K. Scott and G. M. Woods, unpublished). However, it would appear that the intracellular processing pathways differ between neonatal and adult LC, with neonatal LC having a reduced ability to traffic antigen to the cell membrane (D.K. Scott, unpublished).

The neonatal epidermis undergoes developmental processes and these are likely to be in response to the external environment. To assess whether exposure to antigen during the neonatal period altered the development and function of the LC, 1-day-old mice were exposed to the contact sensitizer TNCB and the development and function of the LC network was evaluated. By day 4, MHC-II expression revealed distinctive dendrites compared to untreated mice (Fig. 2).

image

Figure 2.  Epidermal sheets stained for MHC-II from 4-day-old mice. The control represents normal development whereas the TNCB-treated section was prepared from mice exposed to TNCB at birth. Results clearly show that TNCB treatment at birth altered the development of Langerhans cells as evidenced by their more dendritic nature at 4 days, compared with controls.

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This accelerated development translated to an increase in function as antigen applied through this treated skin at 4 days of age did not induce neonatal tolerance and analysis of antigen carriage using the FITC tracking technique revealed that increased levels of antigen reached the draining lymph node. Furthermore, molecules such as Langerin and DEC205 were up-regulated more rapidly.

Consequently, the exposure of the developing epidermis to external stimuli can influence the maturation process and the outcome of the neonatal immune response and have implications in later life. One of these stimuli is UV radiation and although the effects of UV on adult skin have been extensively studied, virtually no attention has been directed toward neonatal skin.

Effects of UVB Radiation on Neonatal Skin

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

To analyze the effects of UV radiation on neonatal skin, we exposed mice on the first day after birth with a single dose of radiation from lamps emitting 40% UVB, the doses ranging from 0.5 to 2 kJ m−2 (58). Development of the LC network was assessed using an antibody against MHC-II. A dose-dependent decrease in epidermal LC density was observed until 4 days post-UV exposure, where the density of LC following the highest dose was reduced to almost zero, and for all doses was below the density present on day 1. Following this depletion, LC density rapidly increased and reached normal levels 10 days post-UVB exposure. At 3 weeks of age, the ability of LC to carry antigen and induce proliferation of T cells was assessed, where it was found that the LC were able to carry more antigen if the mice had been treated with UV radiation compared with control mice. They also induced a greater level of proliferation, and the culture supernatants contained Th2 type cytokines (58).

In subsequent experiments, we are using solar simulated lamps which emit a spectrum closely resembling sunlight. BALB/c mice were irradiated at 3 days of age, using 2–8 kJ m−2 UVB, 2 kJ m−2 approximating the minimum erythemal dose. The LC network after this treatment showed a reduction in LC number compared to controls. The LC density then increased, reaching normal levels at 7 days postirradiation (H. M. McGee and G. M. Woods, in preparation).

To determine whether changes occurred within the lymph nodes and spleens of these mice, these tissues were removed at 3 days and 8 weeks after UV exposure, dissociated into a single cell suspension, labeled with antibodies and assessed by flow cytometry. At 3 days after the UV exposure, there was no difference between the percentage, or number, of cell types within the lymph nodes or spleens of mice treated with solar simulated radiation compared to controls. However, when the lymph nodes were removed 8 weeks after the UV exposure, there was a significant increase in the percentage of T regulatory cells and B cells. No changes were observed in the spleens. For comparison, when adult mice were irradiated with similar doses, no changes at any time point were observed in the spleen or lymph nodes (H. M. McGee and G. M. Woods, in preparation).

The key conclusion from these experiments is that by exposing neonatal skin to a mild erythemal dose of UV causes a gradual increase in the number of Tregs and B cells in the local lymph nodes which becomes significant by adulthood. This altered immune environment would favor antigen specific immune suppression and skin tumor development.

Response of Neonatal Skin to Vitamin D

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

During development there are major changes in the structure and chemical components of the skin as outlined above. One of these is 7-dehydrocholesterol which is found in higher levels in children than in adults (59). The direct action of UVB converts 7-dehydrocholesterol to previtamin D3, which is rapidly transformed by thermoisomeration to form vitamin D3. Hydroxylation steps in the kidney and liver result in the biologically active form of vitamin D3, 1α,25 dihydroxyvitamin D3 (1α,25(OH)2D3) (60). Keratinocytes also have the capacity to complete these enzymatic steps to produce 1α,25(OH)2D3 (61) and along with LC, γδ T cells (62), mast cells, dermal DC and macrophages express the vitamin D receptor (VDR) (63). As such, the production of 1α,25(OH)2D3 within the skin has been proposed to have a role in cutaneous immune responses and UVB-induced immunosuppression (64). Indeed, recent evidence suggests that 1α,25(OH)2D3 alters cutaneous immune responses by acting directly on the cells within the SIS that express the VDR or by acting indirectly via altering keratinocyte production of immunomodulatory cytokines and growth factors.

It is well established that 1α,25(OH)2D3 can influence DC differentiation, maturation and survival and the addition of 1α,25(OH)2D3 to highly enriched LC populations isolated from adult mice reduces MHC-II and costimulatory molecule expression (CD40, CD80 and CD86) (62,65) translating to a reduced ability to stimulate allogenic T cell proliferation (65). In the presence of 1α,25(OH)2D3 in highly purified LC populations there is an increased LC-mediated production of IL-1β and decreased production of IL-10 while tumor necrosis factor-α remains the same. Following LC activation by CD40 ligation increased levels of IL-6 and IL-12p40 were identified (65). The direct action of 1α,25(OH)2D3 on LC can also be influenced via changes in keratinocyte production of immunomodulatory molecules by 1α,25(OH)2D3; again this is likely to differ during the neonatal period. Reduced keratinocyte production of granulocyte-macrophage colony stimulating factor by the addition of 1α,25(OH)2D3 in culture impairs LC maturation and results in a reduced ability to stimulate allogenic T cell proliferation (66). A further mechanism by which 1α,25(OH)2D3 is speculated to alter cutaneous immune responses is via keratinocyte induction of the ligand for receptor activator of nuclear factor-κB (BANKL). This molecule is in part responsible for UVB-mediated immunosuppression by DC and appears to involve the development of Tregs (67). Finally, 1α,25(OH)2D3 augments inflammatory chemokine production by LC and also migratory activity of skin-associated T cells, thereby altering T cell recruitment. Overall in vitro evidence suggests a role for 1α,25(OH)2D3 in the SIS appears to be directed toward a dampening of cutaneous immune responses. In general, in vivo human and mouse data support this concept, as the topical application of a vitamin D analog to human (68) and 1α,25(OH)2D3 to mouse skin (69) prior to the application of a contact sensitizer results in significant immunosuppression, presumably due to its action on LC function.

A further direct action of 1α,25(OH)2D3 and related compounds is to reduce UVB-induced cyclobutane pyrimidine dimers (CPD) within keratinocytes (70,71). Accurate repair of CPD is required, otherwise mutations are introduced. Protection against these mutations is achieved by increasing p53 expression within the nucleus to allow time for DNA repair, but if the DNA damage is irreparable, the cell undergoes apoptosis. The addition of 1α,25(OH)2D3 at physiologically relevant levels in human skin cultures markedly reduces UVR-induced apoptosis. 1α,25(OH)2D3 appears to increase keratinocyte p53 expression while suppressing the nitric oxide pathway which has been shown to inhibit CPD repair (72), thereby increasing survival (70). A further mechanism may be by the induction of metallothionein by 1α,25(OH)2D3 in keratinocytes (73). This antioxidant has been demonstrated in human skin post-UVB exposure (74) and metallothionein knockout mice have increased keratinocyte apoptosis following UVB exposure (75). Thus the production of 1α,25(OH)2D3 may alter factors within keratinocytes to promote the efficient repair of CPD. Further study is required to ascertain whether 1α,25(OH)2D3 is produced in higher levels in children’s skin in response to UVR that will contribute to modulation of DC function and the repair of UVB-induced DNA damage and hence a reduced risk of skin cancer development.

These effects on the SIS and UVB-induced DNA damage led us to investigate the role of dietary vitamin D3 on the development of the SIS in vivo. To this end we developed a vitamin D3-deficient mouse model through dietary and UVB exposure restriction. To investigate DNA damage we exposed neonatal mice to UVB radiation and enumerated the CPD-positive nuclei within the epidermis. Preliminary data suggest that neonatal skin has less DNA damage than adult skin, potentially through structural differences, particularly a thicker layer of keratin. The presence of vitamin D3 within the diet reduced the UVB-induced DNA damage in female, but not male, mice during the neonatal period (Fig. 3).

image

Figure 3.  Examples of immunoperoxidase staining to identify cyclobutane pyrimidine dimers of 4-day-old neonatal skin following exposure to 8 kJ m−2 UVB radiation. The sections convey the information that male neonatal mice exhibit more DNA damage than female neonatal mice and that vitamin D3-deficient mice (both male and female) appear to have more DNA damage than their normal counterparts.

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Implications For Melanoma

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

Animal models to study UV-induced melanoma have been a challenge for many years. Donawho and Kripke (76) showed that UV radiation can serve as the initiator or promoter for the induction of cutaneous melanoma in young (C3H/HeN) mice, although less efficient than DMBA or croton oil. They also found that UV could serve as a cofactor that promotes the growth of melanoma induced by chemical agents. Hence the induction of melanoma is a complex, multifactorial process. They concluded with the question can UV radiation, by itself, induce melanomas in the mouse model?

The answer to this comes from the UV-irradiated HGF/SF transgenic model developed by Frances Noonan et al. (38). When these transgenic mice were irradiated at 3–5 days of age they developed junctional melanomas, which resemble human disease, by 9–12 months. UV irradiation of adult mice was ineffective in producing melanoma, but if given to mice that had received UV irradiation in the neonatal period they developed multiple melanomas. This model provides experimental support for neonatal melanoma susceptibility and evidence that neonatal UV irradiation is sufficient to initiate melanoma (38,77,78). Using this model De Fabo et al. (79) found that UVB is responsible for the induction of cutaneous melanoma whereas UVA is ineffective, even at doses considered physiologically relevant.

Transgenic mouse models are now allowing the exploration of the molecular events involved in melanoma. In cutaneous malignant melanoma in humans INK4a/ARF at 9p21 is a melanoma susceptibility locus with two overlapping tumor suppressor genes, p16 INK4a and p14 ARF (p19 ARF in mice) (77,80). These regulate the cell cycle via pRB and p53 as well as apoptosis and cell senescence (77,81). With human tumors, loss of p16INK4a and with it pRB function are significant mutations (77,82).

The Ras pathway is also pivotal for melanoma development. Hras is reported as a potent amplifier of tumorigenesis in genetically modified mice carrying deletions of the INK4a or ARF genes (81,83). However, treating brown mice (mixed C3H/Sv129) carrying a melanocyte-specific mutant Hras (G12v) transgene (TPras), and using a similar neonatal UVR regime to that used by Noonan et al. (38), Hacker et al. (39) found that Ras activation alone is sufficient to predispose melanocytes to UVR-induced transformation. While the mechanism is unclear, it does not always involve loss of INK4a or ARF. It may promote melanocyte proliferation or interfere with the DNA damage and apoptotic pathways. Further studies from this group in Cdk4 R24C/R24C/ TPras mice show that while Hras activation initiates UVR-induced melanoma development, a cell cycle defect introduced by mutant Cdk4 (inhibits INK4a) contributes to tumor progression producing more aggressive metastatic tumors (84).

How a single neonatal UVR dose is sufficient to induce melanoma in mice carrying Ras or HGF/SF is unclear. It may be that the melanocytes are immature and not fully differentiated and hence neoplastic transformation occurs more readily. There are increased numbers of these cells in the dermis and at the dermal/epidermal junction and so the number of cells prone to UVR injury and transformation is greatly increased. Proliferation may be secondary to differences in melanocyte growth factors, particularly stem cell factor and basic FGF and neurotrophins within neonatal skin that may promote UVB-damaged melanocytes to proliferate or survive. The microenvironment of developing melanoma remains a challenging area for study.

In humans, melanocytic nevi are strong risk factors for the development of melanoma, the latter arising from melanocytes within the nevi (85). Increased sun exposure before the age of 20 is important for the development of nevi, as adolescents with regular high sun exposure have more nevi compared to adolescents with intermittent exposure (86). Whiteman et al. (85) suggested that there are two pathways to melanoma development, one in “nevus-prone people” who progress to melanoma with less sun exposure and the second where melanoma arises after burning doses of UVB radiation. In both human and experimental murine melanoma, while molecular pathways leading to melanoma are being determined, the precise initiating molecular events require definition.

The role of SIS in these neonatal melanoma models may provide important clues to melanoma outcomes in later life. The neonatal environment is prone to tolerance induction and the generation of antigen-specific Tregs, which remain throughout life. Consequently any melanoma antigens generated during the neonatal period are likely to result in the generation of antigen-specific Tregs. As the tumor cells evolve during the adult period these long lived Tregs have the potential to blanket the development of effective antitumor immunity. To us, the difficulty in overcoming this powerful suppressive signal remains a central challenge of current melanoma immunotherapeutic protocols such as melanoma antigen-loaded DC to induce cytotoxic T cells. While cytotoxic T cells can be demonstrated in in vitro systems, frequently patients with metastatic melanoma fail to develop effective antitumor responses, presumably as the cytotoxic T cells are ineffective because of the presence of Tregs.

A further element in neonatal skin that may contribute to neonatal susceptibility to melanoma is a poverty of the inflammatory response. This was first reported 40 years ago by Majno (87). Like Wolnicka-Glubisz et al. (23) we have found that the inflammatory response in neonatal skin fails to develop in response to UV radiation; there is no neutrophil infiltration of the tissue, or vascular dilatation or increased vascular permeability. Further, the neonatal mast cells are enlarged with prominent granules but are not released in response to UV radiation. Normally, in adult responses to UVB radiation there is release, within minutes, of mast cell granules, histamine and other inflammatory mediators (88). This failure to produce an acute inflammatory response limits the development of immunity and promotes a tolerogenic environment.

Conclusion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

The neonatal immune environment and the events that occur during this time have profound effects for the adult period. While positive immune responses/immunity can be developed, the neonatal immune system, particularly the SIS, tends to promote tolerance. For the development of UV radiation-induced melanoma, a single high dose (equivalent to a burning dose in adults) to transgenic mice (HGF/SF or TPras) during the neonatal period results in melanoma. This occurs with a background of UVB-induced immunosuppression and the generation of Tregs. This has implications for the development of human skin cancer, particularly melanoma, where childhood sun exposure is a major risk factor for melanoma on adult life. The UVB-induced immunosuppression and Treg generation in early life may explain the difficulty of producing effective antitumor immunotherapy. Exploring molecular pathways in the neonatal period should lead to future insights into the epidermal microenvironment and SIS, which may provide targets for early disease diagnosis and intervention.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References

Acknowledgements— Dr. Margaret Kripke and colleagues have provided valuable support to various members of our laboratory. One of us (H.K.M.) has spent productive sabbatical periods with Margaret Kripke at the MD Anderson Cancer Center during the 1990s and a number of our graduate students have gained valuable experience and expertise by either working or visiting these laboratories. We would like to acknowledge unpublished data provided by Thanuja Dharmadasa. Research support was provided by the National Health and Medical Research Council, the Cancer Council of Tasmania, the Royal Hobart Hospital Research Foundation and the Arthritis Foundation of Australia.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Neonatal Skin Structure
  5. Response of Neonatal Skin to Antigen
  6. Effects of UVB Radiation on Neonatal Skin
  7. Response of Neonatal Skin to Vitamin D
  8. Implications For Melanoma
  9. Conclusion
  10. Acknowledgments
  11. References
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