Unwelcome guests: macrophages promote UV‐induced melanoma (2011)

The effects of ultraviolet (UV) radiation on skin have long been of clinical and research interest. Ultraviolet radiation can be subdivided into three groups: UVA, UVB, and UVC, where UVA has the longest wavelength. While UVC, the most energetic variety, is blocked by the Earth’s atmosphere, UVA and UVB are not and can impact exposed human skin. Characterization of specific changes induced by UV radiation to endogenous melanocytes and other cellular components of the skin have been hindered by both the complexity of this organ as well as the relative paucity of melanocytes in the skin. To date, description of the effects of UV irradiation on melanocytes has relied heavily on in vitro work. Though these important studies have provided powerful mechanistic insights into UV induced signaling changes, stress responses, and pigment production in melanocytes/skin (reviewed by Abdel-Malek et al., 2010), comprehensive study of specific changes induced by UV on pure populations of melanocytes in their normal physiologic tissue microenvironments has not been possible. To this end, Zaidi et al. (2011) have developed the iDct-GFP transgenic mouse strain, recently described in Nature. iDct-GFP transgenic mice were generated by crossing mice expressing the reverse tetracycline activated transactivator (rtTA) under control of the melanocyte-specific dopachrome tautomerase (Dct) promoter elements with a second strain bearing a histone H2B-GFP fusion protein controlled by the tetracycline response element (TRE). The resulting, iDct-GFP, double transgenic mice rapidly and specifically express GFP in the melanocytic lineage only in the presence of doxycyline administration. In order to study the effects of UV radiation to resident melanocytes under natural physiologic conditions, iDct-GFP mice were perinatally irradiated with UVB. This resulted in increases in both melanocyte number and migration to the epidermis. Irradiated skin was collected and dissociated into a single cell suspension and H2B-GFP expressing melanocytes were purified by fluorescence activated cell sorting. Expression analysis of acutely UVB-irradiated melanocytes (24 h post exposure) confirmed the presence of the predicted stress response including elevated expression of p53 target genes. When melanocytes were purified 6 days after the UVB exposure, an IFN-γ response pattern was observed, which was not seen when analogous experiments were performed in adult mice. In order to address the functional importance of these putative IFN-γ induced expression changes, the investigators performed antibody mediated blockade of IFN signaling in the irradiated mice. Blockade of type II interferon (IFN-γ), but not type I interferons (IFN-α,β) resulted in a reversion of both the melanocyte activation phenotype as well as IFN-γ induced expression alterations to these cells. Of note, IFN-γ signaling blockade also reversed UVB induced expression of the chemokine Ccl8 (MCP-2), a molecule that could be responsible for chemotaxis of an immune cell population that might be producing IFN-γ. An extensive characterization of immune cells present in irradiated skin revealed the presence of an induced population of IFN-γ expressing CD11b+/F4/80+/Gr-1− macrophages. The authors reasoned that these IFN-γ expressing macrophages were very likely to be responsible for inducing the IFN-γ associated changes observed subacutely in melanocytes. Using a combination of mRNA expression analysis, in vitro experiments, and Ccr2 and Ccr5-deficient mice, the authors were able to provide strong support for the hypothesis that local Ccr2-ligand (including Ccl8) was specifically responsible for macrophage chemotaxis following UV exposure. Further, as the arrival of CD11b+/F4/80+ cells coincided with upregulation of Ccl8 in neonatal melanocytes, the authors suggest that a positive feedback loop may develop subacutely in UV irradiated neonatal skin. In this scenario, macrophage IFN-γ production, by stimulating Ccl8 production in melanocytes, induces further macrophage recruitment and IFN-γ stimulation of melanocytes in the irradiated area (Figure 1). UVB irradiation of neonatal mouse skin results in an inflammatory cascade leading to activation of both macrophages and melanocytes. (1) Incoming UVB radiation leads to (2) local Ccr2 ligand release resulting in (3) macrophage recruitment to the skin microenvironment. (4) Subsequent IFN-γ production by the macrophages leads to (5) melanocyte Ccl8 production and further macrophage recruitment/activation in addition to (6) a melanocyte IFN-γ response and upregulation of immune evasion molecules. Macrophages and tumor cells have complex interactions that in many circumstances may facilitate tumor formation and progression. As such, the authors functionally characterized this UV-induced macrophage population with respect to potential pro- or anti-tumor properties. F4/80+ cells were purified from the skin of irradiated neonates and were subsequently admixed with F5061 mouse melanoma cells and then implanted back into syngenic recipient mice. In comparison to a control spleen-derived macrophage population, the UV stimulated population enhanced tumor growth at least in part by decreasing the number of TUNEL-positive (apoptotic) cells. Antibody mediated blockade of IFN-γ prevented this enhancement of tumor growth, suggesting IFN-γ secretion by the macrophages was important in mediating this effect. In order to study the relevance of these findings to human melanomas, the investigators stained a human melanoma tissue microarray for CD68 (a macrophage marker) and IFN-γ. Seventy percent of the melanomas showed positive CD68 staining, all of which also stained positively for IFN-γ. These results demonstrate that CD68+IFN-γ+ cells exist in human melanomas and may have analogous tumor supportive roles to the induced IFN-γ+ macrophage population in the mouse model. In fact, previous studies support this hypothesis, as enhanced macrophage infiltration in melanoma has been shown to correlate with poor survival (Jensen et al., 2009). The role of interactions between the immune system and melanoma cells in tumor formation and progression are very complex. Understanding these interactions and the effects of manipulating them will likely be central to developing more effective melanoma therapeutics. IFN-γ has been used to treat melanoma patients previously, and in support of the authors conclusions, seemed to have a negative impact on patient survival (Meyskens et al., 1995). Recombinant IFNα2b is routinely administered as an adjuvant agent to melanoma patients, with a significant increase in relapse-free survival, but without a significant increase in overall survival (Kirkwood et al., 2004). It should be noted that blockade of type I interferons in the study by Zaidi and colleagues had no detectable phenotypic effect on the mouse model. In this model system, the presence of IFN-γ expressing macrophages was also associated with increases in expression of the negative co-stimulatory molecule Ctla4. Targeting of CTLA4 in addition to other co-stimulatory molecules, such as PD-1 are of much interest in the melanoma field and are emerging as promising new therapies for melanoma patients (Hodi et al., 2010). Further characterization of the interactions between melanoma and the immune system will not only advance our understanding of this disease but may also help to predict which immunomodulatory agents (or combination of agents) are likely to succeed in individual melanoma patients. In addition to elegantly unraveling a complex inflammatory relationship between UV irradiation and its effects on endogenous melanocytes and macrophages, Zaidi and colleagues have also provided an important research tool to the pigment cell and melanoma community. The iDct-GFP mice will allow for unprecedented purification and study of endogenous melanocytes from their normal physiological microenvironment. When combined with other relevant mouse models of melanoma, iDct-GFP mice will aid in more precise study of tumor formation and progression. In addition, these mice will enable new studies on the mechanistic evaluation of complex processes such as metastasis, tumor initiating/stem cell biology, and progression of benign precursor lesions to melanoma.