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4-Tertiary Butyl Phenol Exposure Sensitizes Human Melanocytes to Dendritic Cell-Mediated Killing: Relevance to Vitiligo

2005; Elsevier BV; Volume: 124; Issue: 4 Linguagem: Inglês

10.1111/j.0022-202x.2005.23653.x

1523-1747

Tara M. Kroll, Hemamalini Bommiasamy, Raymond E. Boissy, Claudia Hernandez, Brian J. Nickoloff, Ruben Mestril, I. Caroline Le Poole,

Calpain Protease Function and Regulation

The trigger initiating an autoimmune response against melanocytes in vitiligo remains unclear. Patients frequently experience stress to the skin prior to depigmentation. 4-tertiary butyl phenol (4-TBP) was used as a model compound to study the effects of stress on melanocytes. Heat shock protein (HSP)70 generated and secreted in response to 4-TBP was quantified. The protective potential of stress proteins generated following 4-TBP exposure was examined. It was studied whether HSP70 favors dendritic cell (DC) effector functions as well. Melanocytes were more sensitive to 4-TBP than fibroblasts, and HSP70 generated in response to 4-TBP exposure was partially released into the medium by immortalized vitiligo melanocyte cell line PIG3V. Stress protein HSP70 in turn induced membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and activation of DC effector functions towards stressed melanocytes. Melanocytes exposed to 4-TBP demonstrated elevated TRAIL death receptor expression. DC effector functions were partially inhibited by blocking antibodies to TRAIL. TRAIL expression and infiltration by CD11c+ cells was abundant in perilesional vitiligo skin. Stressed melanocytes may mediate DC activation through release of HSP70, and DC effector functions appear to play a previously unappreciated role in progressive vitiligo. The trigger initiating an autoimmune response against melanocytes in vitiligo remains unclear. Patients frequently experience stress to the skin prior to depigmentation. 4-tertiary butyl phenol (4-TBP) was used as a model compound to study the effects of stress on melanocytes. Heat shock protein (HSP)70 generated and secreted in response to 4-TBP was quantified. The protective potential of stress proteins generated following 4-TBP exposure was examined. It was studied whether HSP70 favors dendritic cell (DC) effector functions as well. Melanocytes were more sensitive to 4-TBP than fibroblasts, and HSP70 generated in response to 4-TBP exposure was partially released into the medium by immortalized vitiligo melanocyte cell line PIG3V. Stress protein HSP70 in turn induced membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and activation of DC effector functions towards stressed melanocytes. Melanocytes exposed to 4-TBP demonstrated elevated TRAIL death receptor expression. DC effector functions were partially inhibited by blocking antibodies to TRAIL. TRAIL expression and infiltration by CD11c+ cells was abundant in perilesional vitiligo skin. Stressed melanocytes may mediate DC activation through release of HSP70, and DC effector functions appear to play a previously unappreciated role in progressive vitiligo. dendritic cell fluorescence activated cell sorting Fas ligand heat shock protein interferon interleukin just another method 4-tertiary butyl phenol tumor necrosis factor TNF-related apoptosis-inducing ligand Vitiligo is an acquired skin disorder, involving an autoimmune response against melanocytes (Boissy, 2001Boissy R.E. Vitiligo.in: Theofilopoulos A.N. Bona C.A. 2nd edn. The Molecular Pathology of Autoimmunity. Gordon and Breach/Harwood Academic Publishers, Langhorne, PA2001: 773-780Google Scholar; Le Poole et al., 2004Le Poole I.C. Wankowicz-Kalinska A. van den Wijngaard R.M.J.G.J. Nickoloff B.J. Das P.K. Autoimmune aspects of depigmentation in vitiligo.J Investig Dermatol Symp Proc. 2004; 9: 68-72Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). It remains to be explained as to what triggers the autoimmune response to melanocytes. Patients frequently refer to skin trauma as an initiating factor for their disease. Melanocyte overexposure to ultraviolet rays may cause deregulation of melanization and/or of mitosis, inducing a stress response in the pigment cell (Jean et al., 2001Jean S. Bideau C. Bellon L. et al.TH expression of genes induced in melanocytes by exposure to 365-nm UVA: Study by cDNA arrays and real-time quantitative RT-PCR.Biochim Biophys Acta. 2001; 1522: 89-96Crossref PubMed Scopus (50) Google Scholar). Sites of mechanical stress will express elevated levels of stress proteins (Kippenberger et al., 1999Kippenberger S. Bernd A. Loitsch S. Muller J. Guschel M. Kaufmann R. Cyclic stretch up-regulates proliferation and heat shock protein 90 expression in human melanocytes.Pigment Cell Res. 1999; 12: 246-251Crossref PubMed Scopus (18) Google Scholar). Burns and cuts have been documented as initiation sites for progressive depigmentation, and the Koebner phenomenon is often observed in vitiligo patients (Le Poole and Boissy, 1997Le Poole C. Boissy R.E. Vitiligo.Seminar Cutan Med Surg. 1997; 16: 3-14Crossref PubMed Scopus (55) Google Scholar). Finally, in individuals sensitive to bleaching phenols, exposure to phenolic compounds in the workplace can cause what has been coined "occupational vitiligo" (Boissy and Manga, 2004Boissy R.E. Manga P. On the etiology of contact/occupational vitiligo.Pigment Cell Res. 2004; 17: 208-214Crossref PubMed Scopus (186) Google Scholar). Skin trauma leads to oxidative stress, and accumulation of H2O2 has been observed in vitiligo lesional skin (Schallreuter et al., 1999Schallreuter K.U. Moore J. Wood J.M. et al.In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase.J Investig Dermatol Symp Proc. 1999; 4: 91-96Abstract Full Text PDF PubMed Scopus (320) Google Scholar). These conditions will induce expression of stress proteins including heat shock protein (HSP)70 and will enhance the activity of anti-oxidative enzymes to protect skin cell viability (Currie and Tanguay, 1991Currie R.W. Tanguay R.M. Analysis of RNA from transcripts for catalase and SP71 in rat hearts after in vivo hyperthermia.Biochem Cell Biol. 1991; 69: 375-382Crossref PubMed Scopus (85) Google Scholar; Calabrese et al., 2001Calabrese V. Sapagnini G. Catalano C. Bates T.E. Geraci D. Pennisi G. Giuffrida Stella A.M. Regulation of heat shock protein synthesis in human skin fibroblasts in response to oxidative stress: Role of vitamin E.Int J Tissue React. 2001; 23: 127-135PubMed Google Scholar; Renis et al., 2003Renis M. Cardille V. Grasso S. Palumbo M. Scifo C. Switching off HSP70 and I-NOS to study their role in normal and H2O2-stressed human fibroblasts.Life Sci. 2003; 26: 757-769Crossref Scopus (11) Google Scholar). In this study, 4-tertiary butyl phenol (4-TBP) was chosen as a model compound to address stress protein expression and its involvement in initiating an autoimmune response to melanocytes by dendritic cells (DC). It has been hypothesized that bleaching compound 4-TBP can serve as an alternative substrate for tyrosinase, which would explain its inhibitory effect on melanin synthesis (Yang and Boissy, 1999Yang F. Boissy R.E. Effects of 4-tertiairy butylphenol on the tyrosinase activity in human melanocytes.Pigment Cell Res. 1999; 12: 237-245Crossref PubMed Scopus (37) Google Scholar). Competitive inhibition of tyrosinase, the rate-limiting enzyme involved in melanogenesis, occurs at low 4-TBP concentrations. Conversion of 4-TBP into semiquinone free radicals can contribute to cellular stress (Boissy and Manga, 2004Boissy R.E. Manga P. On the etiology of contact/occupational vitiligo.Pigment Cell Res. 2004; 17: 208-214Crossref PubMed Scopus (186) Google Scholar). Cytotoxic responses occur at a higher concentration of 4-TBP and are independent of the degree of pigmentation of melanocytes (Yang et al., 2000Yang F. Sarangarajan R. Le Poole I.C. Medrano E.E. Boissy R.E. The cytotoxicity and apoptosis induced by 4-tertiary butylphenol in human melanocytes are independent of tyrosinase activity.J Invest Dermatol. 2000; 114: 157-164Crossref PubMed Scopus (67) Google Scholar). Expression of the A2b receptor for adenosine was enhanced in response to 4-TBP, and expression of this receptor may sensitize melanocytes to apoptosis (Le Poole et al., 1999Le Poole I.C. Yang F. Brown T.L. Cornelius J. Babcock G.F. Das P.K. Boissy R.E. Altered gene expression in melanocytes exposed to 4-tertiary butylphenol (4-TBP): Upregulation of the A2B receptor for adenosine.J Invest Dermatol. 1999; 113: 725-731Crossref PubMed Scopus (25) Google Scholar). Stressed cells are characterized by elevated expression of stress proteins. Stress proteins include the HSP family upregulated in response to elevated environmental temperatures and other forms of stress. Stress proteins are evolutionarily very well conserved, and they function as chaperone molecules protecting cellular proteins from premature degradation by supporting proper protein folding (Houry, 2001Houry W.A. Chaperone-assisted protein folding in the cell cytoplasm.Curr Protein Pept Sci. 2001; 2: 227-244Crossref PubMed Scopus (96) Google Scholar). Cells with elevated levels of stress proteins are protected from the consequences of subsequent stress episodes (Mestril and Dillmann, 1995Mestril R. Dillmann W.H. Heat shock proteins and protection against myocardial ischemia.J Mol Cell Cardiol. 1995; 27: 45-52Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Contrary to the cytoprotective effect of intracellular stress proteins, once released into the extracellular milieu stress proteins can induce an immune response to the very cells from which they were derived. Stress proteins are immunogenic and were shown to serve as antigens in certain autoimmune diseases, which is best explained by the extensive homology observed between human and bacterial stress proteins or "antigen mimicry" (Bell, 1996Bell C.G. Do stress proteins have a regulatory role in autoimmune rheumatoid arthritis?.Biochem Soc Trans. 1996; 24: 494SCrossref PubMed Scopus (2) Google Scholar; Xu, 2003Xu Q. Infections, heat shock proteins and atherosclerosis.Curr Opin Cardiol. 2003; 18: 245-252Crossref PubMed Scopus (39) Google Scholar). Besides serving as antigens, stress proteins also enhance an immune response by inducing phagocytosis and processing of chaperoned antigens by DC (Noessner et al., 2002Noessner E. Gastpar R. Milani V. et al.Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells.J Immunol. 2002; 169: 5424-5432Crossref PubMed Scopus (213) Google Scholar). Consequently, stress proteins have been included as adjuvants in tumor vaccines (Srivastava and Amato, 2001Srivastava P.K. Amato R.J. Heat shock proteins: The 'Swiss army knife' vaccines against cancer and infectious agents.Vaccine. 2001; 19: 2590-2597Crossref PubMed Scopus (149) Google Scholar). Recently, it was reported that DC can specifically kill tumor cells whereas surrounding, healthy cells are left untouched (Janjic et al., 2002Janjic B.M. Lu G. Pimenov A. Whiteside T.L. Storkus W.J. Vujanovic N.L. Innate direct anticancer effector function of human immature dendritic cells. I. Involvement of an apoptosis-inducing pathway.J Immunol. 2002; 168: 1823-1830Crossref PubMed Scopus (83) Google Scholar; Lu et al., 2002Lu G. Janjic B.M. Janjic J. Whiteside T.L. Storkus W.J. Vujanovic N.L. Innate direct anticancer effector function of human dendritic cells. II. Role of TNF, lymphotoxin-alpha(1)beta(2), Fas ligand and TNF-related apoptosis inducing ligand.J Immunol. 2002; 168: 1831-1839Crossref PubMed Scopus (122) Google Scholar). DC-mediated killing was found to be mediated by expression of tumor necrosis factor (TNF) family members on the DC surface, accompanied by the expression of the appropriate receptors by tumor cells (Lu et al., 2002Lu G. Janjic B.M. Janjic J. Whiteside T.L. Storkus W.J. Vujanovic N.L. Innate direct anticancer effector function of human dendritic cells. II. Role of TNF, lymphotoxin-alpha(1)beta(2), Fas ligand and TNF-related apoptosis inducing ligand.J Immunol. 2002; 168: 1831-1839Crossref PubMed Scopus (122) Google Scholar). Healthy control cells do not express the same levels of such receptors, and are thus protected from DC-mediated killing (Lu et al., 2002Lu G. Janjic B.M. Janjic J. Whiteside T.L. Storkus W.J. Vujanovic N.L. Innate direct anticancer effector function of human dendritic cells. II. Role of TNF, lymphotoxin-alpha(1)beta(2), Fas ligand and TNF-related apoptosis inducing ligand.J Immunol. 2002; 168: 1831-1839Crossref PubMed Scopus (122) Google Scholar). The hypothesis under study is that DC are equally capable of killing stressed melanocytes to initiate an autoimmune response resulting in progressive depigmentation of the skin. The direct effects of 4-TBP exposure on cell viability of control and vitiligo-derived melanocytes was measured. Induction of HSP70 induction was assessed, and expression of HSP70 was artificially elevated by adenoviral overexpression to evaluate its cytoprotective effect. DC exposed to activating stress proteins or activated by interferon-γ (IFN-γ) were reacted with stressed and unstressed melanocytes, and resulting melanocyte death was measured. The cytotoxicity observed was correlated to membrane expression of TNF family members by DC, and to corresponding death receptors on stressed melanocytes. Finally, the results were correlated to observations in vitiligo skin by immunohistology. These studies were performed to evaluate a possible role of stress proteins and of DC in initiating depigmentation. In Figure 1, the viability of normal melanocyte culture Mc0009 P12, as well as immortalized cell lines PIG1 and PIG3V, and normal fibroblast culture Ff9929 P7 was shown in the presence or absence of 4-TBP. At relatively low concentrations of 4-TBP (250 μM), the viability of both immortalized cell lines, PIG1 and particularly PIG3V, was significantly reduced (to 59.1% and 37.5%, respectively). The difference in viability among PIG1 and PIG3V cells was not considered significant at p=0.11 in a t test. The viability of primary fibroblast and melanocyte cell cultures was not affected at 250 μM of 4-TBP. Overall, fibroblasts were less sensitive to 4-TBP than melanocytes and a significant reduction in fibroblast viability was noted only at 1 mM of 4-TBP (p=0.001). Expression of HSP70 by immortalized melanocytes cultured in the presence or absence of 4-TBP is shown in Figure 2a. It can be observed that the level of intracellular HSP70 increased up to 6.1-fold in PIG1 control melanocytes and 5.2-fold in PIG3V vitiligo melanocytes in the presence of 4-TBP when compared with untreated cells. Interestingly, a 3.3-fold increase in the release of HSP70 was also observed for PIG3V vitiligo melanocytes following treatment with 500 μM 4-TBP as shown in Figure 2b. Moreover, a 5.3-fold increase in the HSP70 content of the medium was noted for PIG3V versus PIG1 melanocytes, further supporting that the vitiligo melanocytes secrete a relatively larger proportion of the stress proteins. Melanocytes overexpressing HSP27 or HSP70 were treated with 4-TBP in the range of 0-1000 μM for 72 h prior to measuring cell viability. Adenoviral overexpression of HSP70 by melanocytes following adenoviral infection was confirmed by western blotting as shown in Figure 3. A 3.7-fold increase in HSP70 content was demonstrated only for cells exposed to AdHSP70, with no increase observed following exposure to other adenoviruses. Western blot analysis of HSP27 expression revealed that the stress of the adenoviral infection procedure per se upregulated HSP27 expression to a similar extent in all three samples compared with untreated cells (not shown). Similar results were observed for PIG1 cells (not shown). As shown in Figure 4, it was observed that adenoviral overexpression of either HSP27 or HSP70 did not adequately protect the cells from 4-TBP-induced cell death at any of the concentrations tested. The same results were obtained when testing PIG3V, demonstrating that a lack of protection by stress proteins also occurred in vitiligo cells (results not shown).Figure 4Lack of protection from apoptosis by heat shock proteins (HSP). Cell viability was measured by trypan blue exclusion of transfected Mc0009 P12 melanocytes following exposure to 4-tertiary butyl phenol (4-TBP) for 72 h. Overexpression of either HSP27 or HSP70 did not protect melanocytes from 4-TBP-induced cell death at any of the concentrations tested.View Large Image Figure ViewerDownload (PPT) In Figure 5, the cytotoxicity of DC toward normal melanocytes and immortalized PIG1 cells is shown. Normal melanocyte culture Mf0201 P5 was pretreated with or without 250 μM 4-TBP for 24 h. DC were either immature DC or cells activated in the presence of 1 μg per mL of HSP 27, 60, and 70 for 48 h. Pre-treatment of DC with HSP clearly activated the cytotoxic ability of the DC, increasing cell death for both target cell types, most notably for melanocytes exposed to 4-TBP (from 7.4% to 65.2%). Melanocytes cultured in the presence of 4-TBP were sensitized to killing by HSP-activated DC, increasing the cytotoxicity 5.8-fold when chromium release was measured after 48 h compared with cells not treated with the bleaching agent. In Figure 6, upregulation of TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 (TRAILR1 and TRAILR2), and TNF receptors 1 and 2 (TNFR1 and TNFR2) was shown after exposing melanocytes to 4-TBP. The mean fluorescence intensities were increased to 8.5-, 6.3-, 1.8-, and 2.9-fold over untreated cells, respectively, in the presence of 4-TBP, suggesting a potential role in sensitizing melanocytes to DC-mediated killing. Meanwhile, Fas expression was reduced to 0.6-fold at 250 μM 4-TBP. Fluorescence activated cell sorting (FACS) histograms also show upregulation of the HSP receptor CD91 (1.7-fold) and more so of tyrosinase-related protein (TRP)-1 (2.2-fold) at 125 μM 4-TBP. Finally, suppression of stem cell factor receptor c-KIT was observed for both 4-TBP concentrations, reducing expression to 0.3-fold at 250 μM 4-TBP. A 2.2-fold increase in the mean fluorescence intensity for TRAIL expression by IFN-γ treated DC was shown in Figure 7. TNF and Fas ligand (FasL) expressions were not upregulated by IFN-γ on the DC membrane. Similar upregulation of TRAIL expression was observed by DC exposed to a cocktail of HSP 27, 60, and 70 (not shown). Histograms representing TRAIL expression in the absence and presence of 4-TBP demonstrate that 4-TBP exposure cannot directly mediate TRAIL upregulation by DC (not shown). Melanocytes were exposed to soluble killer TRAIL or to activated DC, in the presence and or absence of TRAIL-reactive antibodies as shown in Figure 8. Killer TRAIL was cytotoxic for up to 37% of PIG1 melanocytes, and this effect was negated in the presence of the anti-TRAIL Ab RIK-2, which restored viability to 95%. More killing of PIG1 cells was observed in the presence of activated DC (53%) than by soluble killer TRAIL, and this response was only partially inhibited by RIK-2, restoring viability to 62%. This suggests that other mechanisms in addition to TRAIL contribute to melanocyte killing by DC. An increase in the number of infiltrating CD11c+ DC was observed in perilesional skin (78.03 cells per mm epidermis) when compared with with non-lesional skin from the same patients (37.35 cells per mm epidermis). This difference was found to be significant in a one-tailed paired t test (n=4; p=0.026). It should be noted that it was not known whether these patients were exposed to bleaching phenols prior to the onset of their vitiligo. In Figures 9a and b, perilesional skin sections were shown with representative examples of focal infiltration CD11c+ and TRAIL+ cells, respectively. In Figure 9c, co-detection of CD11c (red) and TRAIL (blue) is depicted. Examples of cells co-expressing both markers are highlighted by purple arrows. These results suggested that TRAIL-expressing DC specifically infiltrate perilesional skin of vitiligo patients with active disease. Expression of TRAILR1 was confined to cells in the basal layer of the epidermis directly adjacent to the border of the lesion. An example of gp100+ melanocytes (blue) expressing TRAILR1 (red) is shown in Figure 9d. Expression was typically observed in a region spanning up to ten melanocytes directly adjacent to the lesion. It is possible that some of the TRAILR1 expression observed should be assigned to interspersing keratinocytes; however, keratinocytes are expected to be less affected by TRAILR1 expression than melanocytes because of their high turnover rate. Expression of TRAILR2 was not observed in vitiligo perilesional skin (not shown). Topically applied phenolic compounds, including 4-TBP, can selectively damage melanocytes in the basal layer of the epidermis and can lead to progressive skin depigmentation in some individuals. At higher concentrations (>100 μM), 4-TBP is cytotoxic to melanocytes more so than to keratinocytes (Yang et al., 2000Yang F. Sarangarajan R. Le Poole I.C. Medrano E.E. Boissy R.E. The cytotoxicity and apoptosis induced by 4-tertiary butylphenol in human melanocytes are independent of tyrosinase activity.J Invest Dermatol. 2000; 114: 157-164Crossref PubMed Scopus (67) Google Scholar). PIG3V vitiligo melanocytes showed a tendency to be more sensitive to 4-TBP than control melanocyte cell line PIG1. The putative cytoprotective effect of stress proteins was inadequate to prevent cell death in either cell line as adenoviral overexpression of either HSP27 or HSP70 failed to protect PIG1 or PIG3V melanocytes from 4-TBP-induced cell death. Such a lack of protection can possibly be explained by inadequate activation of antioxidant enzymes, as supported by the therapeutic potential of pseudocatalase in vitiligo (Schallreuter et al., 1999Schallreuter K.U. Moore J. Wood J.M. et al.In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase.J Investig Dermatol Symp Proc. 1999; 4: 91-96Abstract Full Text PDF PubMed Scopus (320) Google Scholar). Both the PIG1 and PIG3V cell lines upregulated HSP70 expression in response to 4-TBP, and vitiligo melanocytes released a larger proportion of HSP70 into the media. HSP70 released into the medium is likely the result of active secretion by vitiligo melanocytes, as (a) increased release by vitiligo melanocytes over control melanocytes was observed at non-toxic 4-TBP concentrations and (b) HSP70 release was increased by vitiligo melanocytes but not control melanocytes at 500 μM 4-TBP, a concentration that was equally cytotoxic to both cell types. It is of interest that this observation is paralleled by less homogeneous HSP70 immunostaining in perilesional epidermis from three vitiligo patients than in non-lesional skin from the same individuals (not shown), suggesting that release of HSP70 may occur from progressive vitiligo epidermis in vivo. Release of HSP70 from viable cells was reported for the constitutive as well as the inducible form of HSP70 (Barreto et al., 2003Barreto A. Gonzalez J.M. Kabingu E. Asea A. Fiorentino S. Stress-induced release of HSC70 from human tumors.Cell Immunol. 2003; 222: 97-104Crossref PubMed Scopus (105) Google Scholar; Broquet et al., 2003Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release.J Biol Chem. 2003; 278: 21601-21606Crossref PubMed Scopus (244) Google Scholar). The mechanism reportedly involves the release of membrane-bound HSP70 from lipid rafts (Broquet et al., 2003Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release.J Biol Chem. 2003; 278: 21601-21606Crossref PubMed Scopus (244) Google Scholar). For the constitutive form of HSP70, it was demonstrated that release was inducible by a variety of cytokines, most notably IFN-γ (Barreto et al., 2003Barreto A. Gonzalez J.M. Kabingu E. Asea A. Fiorentino S. Stress-induced release of HSC70 from human tumors.Cell Immunol. 2003; 222: 97-104Crossref PubMed Scopus (105) Google Scholar). The latter cytokine is generated in perilesional vitiligo skin by infiltrating T cells (Le Poole et al., 2003Le Poole I.C. Stennett L.S. Bonish B.K. et al.Expansion of vitiligo lesions is associated with reduced epidermal CDw60 expression and increased expression of HLA-DR in perilesional skin.Br J Dermatol. 2003; 149: 739-748Crossref PubMed Scopus (27) Google Scholar; Wankowicz-Kalinska et al., 2003Wankowicz-Kalinska A. van den Wijngaard R.M. Tigges B.J. et al.Immunopolarization of CD4+ and CD8+ T cells to type-1 like is associated with melanocyte loss in human vitiligo.Lab Invest. 2003; 83: 683-695Crossref PubMed Scopus (169) Google Scholar). Exposure to 4-TBP enhanced HSP70 expression by melanocytes. Stress proteins occasionally secreted by viable cells have been coined "chaperokines", reflecting the intercellular effects commonly assigned to cytokines while chaperoning peptides specific to the cell type from which they were derived (Asea et al., 2000Asea A. Kabingu E. Stevenson M.A. Calderwood S.K. HSP70 peptide bearing and peptide-negative preparations act as chaperokines.Cell Stress Chaperones. 2000; 5: 425-431Crossref PubMed Scopus (123) Google Scholar; Asea, 2003Asea A. Chaperokine-induced signal transduction pathways.Exerc Immunol Rev. 2003; 9: 25-33PubMed Google Scholar). This may reflect an early phase of an immune response, as HSP70 was shown to induce secretion of primary cytokines IL-1, IL-6, and TNF-α by monocytes/macrophages in a C14-dependent fashion (Asea et al., 2000Asea A. Kabingu E. Stevenson M.A. Calderwood S.K. HSP70 peptide bearing and peptide-negative preparations act as chaperokines.Cell Stress Chaperones. 2000; 5: 425-431Crossref PubMed Scopus (123) Google Scholar). It was previously shown that melanocytes can generate primary cytokines as well (Kruger-Krasagakes et al., 1995Kruger-Krasagakes S. Krasagakis K. Garbe C. Diamantstein T. Production of cytokines by human melanoma cells and melanocytes.Recent Results Cancer Res. 1995; 139: 155-168Crossref PubMed Scopus (37) Google Scholar). As the FACS data shown in Figure 6 support upregulated expression of HSP receptor CD91 by melanocytes in response to 4-TBP, cytokine release from melanocytes may be indirectly increased following 4-TBP exposure. Following induction of an innate immune response, stress proteins induce antigen-specific immunity by enhancing antigen uptake and processing by DC (Noessner et al., 2002Noessner E. Gastpar R. Milani V. et al.Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells.J Immunol. 2002; 169: 5424-5432Crossref PubMed Scopus (213) Google Scholar). In this study it was demonstrated that HSP likewise induce DC effector functions. Similarly, HSP70 was shown to enhance natural killer cell cytolytic activity (Multhoff et al., 1999Multhoff G. Mizzen L. Winchester C.C. et al.Heat shock protein 70 (Hsp70) stimulates proliferation and cytolytic activity of natural killer cells.Exp Hematol. 1999; 27: 1627-1636Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Results obtained with purified HSP must be considered with some caution as commercial preparations continue to improve in purity, and the effects of HSP on DC activation have been assigned to contaminants (in particular lipopolysaccharide) by others (Bausinger et al., 2002Bausinger H. Lipsker D. Ziylan U. et al.Endotoxin-free heat shock protein 70 fails to induce APC activation.Eur J Immunol. 2002; 32: 3708-3713Crossref PubMed Scopus (203) Google Scholar). Commercially obtained purified low-endotoxin HSP used in this study contained<50 EU per mg endotoxin. In accordance with earlier reports, IFN-γ was also shown to activate DC effector functions (Fanger et al., 1999Fanger N.A. Maliszewski C.R. Schooley K. Griffith T.S. Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis inducing ligand (TRAIL).J Exp Med. 1999; 190: 1155-1164Crossref PubMed Scopus (336) Google Scholar). Cell surface expression of different TNF family molecules has been implicated in DC effector functions (Lu et al., 2002Lu G. Janjic B.M. Janjic J. Whiteside T.L. Storkus W.J. Vujanovic N.L. Innate direct anticancer effector function of human dendritic cells. II. Role of TNF, lymphotoxin-alpha(1)beta(2), Fas ligand and TNF-related apoptosis inducing ligand.J Immunol. 2002; 168: 1831-1839Crossref PubMed Scopus (122) Google Scholar). Our results indicate that TRAIL is a major player in DC-mediated cytotoxicity toward stressed melanocytes. This is supported by previous reports of a crucial role for TRAIL in DC-mediated killing of tumor cells (Fanger et al., 1999Fanger N.A. Maliszewski C.R. Schooley K. Griffith T.S. Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis inducing ligand (TRAIL).J Exp Med. 1999; 190: 1155-1164Crossref PubMed Scopus (336) Google Scholar). Here, DC activation was accompanied by enhanced expression of TRAIL, but not of TNF or FasL by DC. Direct exposure to 4-TBP did not induce TRAIL expression by DC (not shown). Exposure of target melanocytes to 4-TBP did induce expression of TRAIL receptors as well as TNF-α receptors, but not Fas. Enhanced receptor expression sensitizes melanocytes to DC-mediated killing, as observed in cytotoxicity assays. TRAIL expressing DC could be replaced at least in part by soluble killer TRAIL, supporting a contribution for TRAIL in melanocyte cytotoxicity. DC-mediated cytotoxicity is commonly thought to be restricted to tumor cells, whereas normal tissue cells remain unaffected. It has been shown, however, that certain preparations of soluble TRAIL are less selective toward malignantly transformed cells (Qin et al., 2001Qin J. Chaturvedi V. Bonish B.