Generalized vitiligo in humans has been correlated with the presence of CD8 T cells specific for melanocyte differentiation antigen in perilesional skin and blood (4
). In the present study, we have characterized a murine CD8 T cell dependent autoimmune vitiligo model with features resembling the generalized human disease. Vitiligo in this model is slowly progressive, with juvenile lesions forming bilaterally in head and facial areas, and only arising later in the body of adult animals. Development of vitiligo is entirely dependent on CD8 T cells, while CD4 T cells exert a negative regulatory effect. Thus, it differs significantly from another model involving TCR transgenic expressing CD8 T cells specific for another melanocyte differentiation protein, gp100. This TCR was derived from a gp100+ mouse, and vitiligo development depends on both CD4 T cells and genetic deficiency of CTLA-4 (47
). It also differs from another TCR transgenic model involving CD4 T cells specific for TRP-1, in which vitiligo fails to develop because a relatively low number of TCR transgenic T cells are efficiently deleted (48
). However, disease can be induced upon transfer into sublethally irradiated recipients, as we demonstrate here. Our model is most similar to one involving mice expressing lysozyme under the control of the TRP-2 promoter in conjunction with a class II MHC-restricted transgenic TCR, in which disease develops spontaneously with what appears to be a similar temporal and spatial progression, but does not initiate for 3 weeks (23
The ability to quantify disease progression over time and in different locations allowed inquiry into the factors contributing to depigmentation. Our work demonstrates a strong direct association of vitiligo with a local increase in CD8 T cells in actively depigmenting perilesional areas relative to adjacent non-pigmented or fully depigmented areas. In addition, the impaired development of vitiligo in CXCR3-/-
mice was associated with greatly diminished infiltration of CD8 T cells into skin. CD8 effectors normally acquire CXCR3, and this chemokine receptor has been shown to mediate infiltration into a variety of inflamed sites (35
). These results are consistent with a model in which the temporal and spatial pattern of depigmentation develops in association with localized inflammatory processes that result in the expression of CXCR3 ligands, and lead to the recruitment of CD8 T cells into the skin. However, this simple model does not readily account for other observations in these mice. One exceptional site is the ears, which depigment normally in juvenile CCR5-/-
FH mice. Ablation of these chemokine receptors does not alter the normal representation of CD8 T cells in the ears of these mice, suggesting that alternate receptors are employed. In keeping with this, adult animals allowed to regain CD8 T cells after sustained depletion from birth show minimal CD8 infiltration and no evidence of ear depigmentation, suggesting that the receptors or ligands mediating ear infiltration are developmentally regulated.
The rapid development of vitiligo in facial and head areas of juveniles demonstrates the presence of immunological effector mechanisms with the potential to cause complete depigmentation. However, there is also a significant infiltration of CD8 T cells into nonlesional skin and hair follicles in juveniles, yet these latter areas do not begin to depigment for 2 months. Conversely, body hair and facial areas depigment simultaneously in adult animals allowed to regain CD8 T cells after sustained depletion from birth. It has been suggested that the development of vitiligo reflects a differential susceptibility of melanocytes to immune mediated destruction (49
). Thus, it is possible that body hair melanocytes in juveniles are less susceptible to immune destruction than those of the head and body areas. Interestingly, juvenile pattern depigmentation is partially blocked by ablation of either CCR5 or CXCR3, while adult pattern depigmentation is only sensitive to ablation of CXCR3. The strong dependence of CD8 T cell infiltration into juvenile skin on CXCR3 suggests that CCR5 plays a different role. CCR5 signaling enhances the activation of both CD8 T cells (50
) and macrophages (51
). This suggests that there are differences in the quality or activation state of immunological effectors infiltrating these distinct body areas as well. While further work will be required to distinguish these possibilities, our results point to factors over and above simple CD8 T cell infiltration that control vitiligo development.
Our work also provides some insight into the mechanisms responsible for melanocyte destruction. It was recently shown that perilesional CD8 T cells directly destroyed melanocytes in a skin explant model through an apoptotic mechanism (8
), but the mode of induction was not defined. In a transgenic model involving CD4 T cell recognition of melanocyte expressed antigen, it was shown that vitiligo development was partially dependent upon expression of Fas-ligand (23
). We found that perforin was not necessary for normal lesion development, with the exception of incomplete depigmentation of epidermis surrounding the eyes and the muzzle, although we cannot exclude a redundant contribution of this pathway to melanocyte loss. However, we found IFNγ to be essential. This is consistent with earlier work showing that transgenic expression of IFNγ in murine skin results in accumulation of T lymphocytes and macrophages into the epidermis, reduced numbers of melanocytes in the hair follicles and hypopigmentation, and in some animals, alopecia (53
The mechanism of action of IFNγ in relation to vitiligo development is likely to be multifaceted. IFNγ expression can enhance CD8 T cell infiltration into skin in multiple ways, including upregulation of CXCR3 (39
), the induction of CXCR3 ligands in peripheral tissue (40
), and increased expression of ICAM-1 on endothelial cell surfaces (54
). The reduced number of CD8 T cells infiltrating adult skin of both CXCR3-/-
mice is consistent with a model in which activation of resident CD8 T cells in the skin leads to local expression of IFNγ, and enhanced expression of ICAM-1 and CXCL9, 10, and 11. This would lead to the recruitment of additional CD8 T cells. However, the diminished CD8 T cell representation is evident in nonlesional skin of both juveniles and adults. This suggests that there are at least two distinct IFNγ dependent processes occurring in these mice: one leading to steady-state infiltration of CD8 T cells into skin, and another leading to augmented infiltration, perhaps in association with more fulminant effector function. Only this latter process leads to vitiligo development. An important, but unresolved issue, is whether this latter process is due to local activation of CD8 T cells already present in skin, or to activation of CD8 T cells in the draining LN that then enter the skin in a CXCR3 and IFNγ –dependent manner. In keeping with this, most T cells in FH AAD+
mice show no evidence of ongoing activation (unpublished), presumably because of limited availability of antigen or APC. Thus, we hypothesize that the development of vitiligo is also dependent on changes in the presentation of tyrosinase that lead to immunogenic rather than tolerogenic T cell activation.
Our data also suggests that IFNγ may play a more direct role in melanocyte destruction apart from its influence on CD8 T cell infiltration. First, vitiligo induced by adoptive transfer of FH T cells into sublethally irradiated recipients is diminished when those cells do not express IFNγ. More directly, CD8 T cell infiltration into juvenile ears was not influenced in either CXCR3-/-
mice. However, while vitiligo developed normally in the ears of CXCR3-/-
mice, it was significantly impaired in those lacking IFNγ. In several models, IFNγ has been shown to directly induce apoptosis (55
), and to enhance expression of Fas (57
). IFNγ also supports the recruitment and activation of macrophages (42
). Activated macrophages produce TNFα, which has previously been shown to induce apoptosis of hair bulb melanocytes (60
). Thus, we propose that the contribution of IFNγ to vitiligo development is two fold: it promotes the trafficking of melanocyte-specific CD8 T cells and macrophages to the skin and participates directly or indirectly in melanocyte destruction.
Overall, this report has demonstrated that autoimmune vitiligo is a complex, temporally and spatially determined process. Importantly, CD8 T cells can be pervasively present in the skin in the steady state without inducing vitiligo in most areas. This points to developmental differences in melanocyte susceptibility and/or immunological effector mechanisms over time, or in different body locations. We show that vitiligo development is strongly dependent on both IFNγ and CXCR3, while dependence on CCR5 is more limited, and both CCR4 and perforin are dispensable. Our results suggest that antagonists against IFNγ or CXCR3 during lesion expansion periods may serve to limit T cell influx to the skin and preserve pigmentation.