Immune responses in a mouse model of vitiligo with spontaneous epidermal de- and repigmentation.

To generate a mouse model of spontaneous epidermal depigmentation, parental h3TA2 mice, expressing both a human-derived, tyrosinase-reactive T-cell receptor on T cells and the matching HLA-A2 transgene, were crossed to keratin 14-promoter driven, stem cell factor transgenic (K14-SCF) mice with intra-epidermal melanocytes. In resulting Vitesse mice, spontaneous skin depigmentation precedes symmetrical and sharply demarcated patches of graying hair. Whereas the SCF transgene alone dictates a greater retinoic acid receptor-related orphan receptor gamma (RORγt)(+) T-cell compartment, these cells displayed markedly increased IL-17 expression within Vitesse mice. Similar to patient skin, regulatory T cells were less abundant compared with K14-SCF mice, with the exception of gradually appearing patches of repigmenting skin. The subtle repigmentation observed likely reflects resilient melanocytes that coexist with skin-infiltrating, melanocyte-reactive T cells. Similar repigmenting lesions were found in a different TCR transgenic model of vitiligo developed on an SCF transgenic background, supporting a role for SCF in repigmentation.

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy, and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus, whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.

A central role for inducible heat-shock protein 70 in autoimmune vitiligo.

Inducible heat-shock protein 70 (HSP70i) is a protein regulated by stress that protects cells from undergoing apoptosis. Such proteins are marvellously well conserved throughout evolution, which has placed them in the spotlight for helping to understand the intriguing relationship between infection and immunity. In the presence of stress proteins, dendritic cells (DCs) will sense this alarm signal and respond by recruiting immune cells of different plumage to fit the occasion. In times of stress, melanocytes will secrete antigen-bound HSP70i to act as an alarm signal in activating DCs that comes equipped with an address of origin to drive the autoimmune response in vitiligo. Here we pose that if the autoimmune response is funnelled through HSP70i, then blocking the stress protein from activating DCs can lend new treatment opportunities for vitiligo.

Mutant HSP70 reverses autoimmune depigmentation in vitiligo.

Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell-mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

HSP70i is a critical component of the immune response leading to vitiligo.

HSP70i and other stress proteins have been used in anti-tumor vaccines. This begs the question whether HSP70i plays a unique role in immune activation. We vaccinated inducible HSP70i (Hsp70-1) knockout mice and wild-type animals with optimized TRP-1, a highly immunogenic melanosomal target molecule. We were unable to induce robust and lasting depigmentation in the Hsp70-1 knockout mice, and in vivo cytolytic assays revealed a lack of cytotoxic T-lymphocyte activity. Absence of T-cell infiltration to the skin and maintenance of hair follicle melanocytes were observed. By contrast, depigmentation proceeded without interruption in mice lacking a tissue-specific constitutive isoform of HSP70 (Hsp70-2) vaccinated with TRP-2. Next, we demonstrated that HSP70i was necessary and sufficient to accelerate depigmentation in vitiligo-prone Pmel-1 mice, accompanied by lasting phenotypic changes in dendritic cell subpopulations. In summary, these studies assign a unique function to HSP70i in vitiligo and identify HSP70i as a targetable entity for treatment.

Characterization of lymphocyte subsets over a 24-hour period in Pineal-Associated Lymphoid Tissue (PALT) in the chicken.

BACKGROUND: Homeostatic trafficking of lymphocytes in the brain has important relevance to the understanding of CNS disease processes. The pineal gland of the chicken contains large accumulations of lymphocytes that suggest an important role related to homeostatic circadian neuro-immune interactions. The purpose of this initial study was to characterize the lymphocyte subsets in the pineal gland and quantitate the distribution and frequency of lymphocyte phenotypes at two time points over the 24-hour light:dark cycle. RESULTS: PALT comprised approximately 10% of the total pineal area. Image analysis of immunocytochemically stained sections showed that the majority of lymphocytes were CD3+ (80%) with the remaining 20% comprising B-cells and monocytes (Bu-1+), which tended to distribute along the periphery of the PALT. T-cell subsets in PALT included CD4+ (75-80%), CD8+ (20-25%), TCRalphabeta/Vbeta1+ (60%), and TCRgammadelta+ (15%). All of the T-cell phenotypes were commonly found within the interfollicular septa and follicles of the pineal gland. However, the ratios of CD8+/CD4+ and TCRgammadelta+/TCRalphabeta/Vbeta1+ within the pineal tissue were each 1:1, in contrast to the PALT where the ratios of CD8+/CD4+ and TCRgammadelta+/TCRalphabeta/Vbeta1+ each approximated 1:4. Bu-1+ cells were only rarely seen in the pineal interstitial spaces, but ramified Bu-1+ microglia/macrophages were common in the pineal follicles. Effects of the 24-h light:dark cycle on these lymphocyte-pineal interactions were suggested by an increase in the area of PALT, a decline in the density of TCRalphabeta/Vbeta1+ cells, and a decline in the area density of Bu-1+ microglia at the light:dark interphase (1900 h) compared to the dark:light interphase (0700 h). CONCLUSION: The degree of lymphocyte infiltration in the pineal suggests novel mechanisms of neuro-immune interactions in this part of the brain. Our results further suggest that these interactions have a temporal component related to the 24-hour light:dark cycle and that CD8+ and TCRgammadelta+ T-cells are preferentially recruited to the pineal follicles. Pineal microglia/macrophages were common and represent an important candidate for mediating these lymphocyte-pineal interactions via secretion of cytokines and chemokines.