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Microscopic changes in vitiligo
ELSEVIER
Microscopic
Changes in Vitiligo
I. CAROLINE LE POOLE, PRANAB K. DAS, PhD
V
PhD
itiligo is characterized by acquired depigmentation in expanding lesions of the skin.] Microscopic ’ analysis has contributed greatly to our present level of understanding of the etiopathology of vitiligo. Light microscopy has been used to reveal changes in the skin associated with depigmentation at the cellular level. Electron microscopy has multiplied the level of magnification roughly by a factor of 100, useful to examine more intimate details at the subcellular level. Combining microscopy with the use of labeled antibodies, the resolution of microscopy has been raised to the molecular level, so that the presence or absence of specific antigens can easily be followed under a variety of circumstances. These methods have provided a means to examine the physiology of melanocytes in conjunction with various components of the tissue environment. Microscopic analysis is of particular use for enigmatic diseasessuch as vitiligo, where a comparison of diseased versus healthy tissue can further direct research concerning the pathomechanism of the disease. Results from these studies have implemented functional assays that use various model systems, including monocultures of melanocytes, cocultures, skinequivalent models, organotypic culture of human skin explants, and animal models.2-’ The development of effective treatment is dependent on an understanding of the events leading to depigmentation in vitiligo. This marks the importance of investigations undertaken to determine whether dormant melanocytes remain within the depigmented lesions, or whether loss of pigmentation involves loss of pigment ceils altogether. Using in vitro cultured cells, the physiology of melanocytes from control and from vitiligo donors has been compared within a defined and adjustable environment. Using cultured cells, evidence has been acquired for potential intrinsic abberancies of vitiligo melanocytes. It must be borne in mind however, that depigmentation in vivo may ultimately be brought
about by environmental factors. In this respect, surrounding keratinocytes may generate a hostile environment for melanocytes. Within a hostile environment, the pigment cell is particularly vulnerable because of its low turnover rate. The histology of perilesional as well as nonlesional vitiligo skin can be informative as to the physiology of melanocytes as well as to cells and matrix molecules comprising the direct environment of the pigment cell. It is conceivable that, at the clinical level, melanocytes appear to be selectively affected, whereas in actual fact, the environment is altered as well. Immunohistochemical evidence is accumulating to show that cellular immunity is involved in vitiligo. In addition to humoral immunity as discussed in the previous chapter, T cells and macrophages are potentially involved in the process of depigmentation. The mode of pigment elimination proposed here would suggest vitiligo to be the effective variant of an in situ immune reaction that is active, but ineffective, in melanoma. The outcome of in viva and in vitro studies appears to support the view that vitiligo is
Loss of Melanocytes
in Vitiligo
Skin
Depigmentation of the skin is potentially a consequence of either of two conditions: (1.) melanocytes may be absent from depigmented skin, or (2) melanogenesis may have been silenced in melanocvtes still present within the lesion. Examples of the absence of melanocytes in depigmented lesions exist in piebaldism, where melanocytes do not populate all areas of the skin during development as a consequence of mutations in the (.-KIT stem cell factor receptor and in Waardenburg syndrome types I-III, where focal absence of melanoeytes appears to be a consequence of mutations in the PAX or MITF transcription factor genes.y 1’) In contrast. silenced mel-
anogenesis is found in albinism, where pigmentation either is reduced or is totally absent as a consequence of mutations in pigmentation-associated genes, to an extent dependent on the type of mutation as well as on the pigmentary gene affected. 11-13 Reduced pigmentation indicates that melanocytes are still present, as in oculocutaneous albinism (OCA) types II and III. It follows that when pigmentation is totally absent from the lesions as in vitiligo, the presence or absence of melanocytes remains an open question. Occasionally, melanocyte cultures can be initiated from lesional vitiligo skin (personal observation); moreover, it has been reported that residual tyrosinase activity remains in vitiligo lesions. 14 As activity of this enzyme is solely restricted to melanocytes, it appears that at least a subpopulation of melanocytes remains present within the lesion. Dormant melanocytes can potentially be detected by histological analysis at the light microscopic (LM) or electron microscopic (EM) level.r5J6 It has been reported that neither histology nor electron microscopic investigation of lesional skin revealed the presence of any ‘clear cells’ within the lesion;lT-I9 however, it can be argued that certain melanocytic properties change along with the observed loss of melanization. Presence or absence of melanocytes in vitiligo lesions has, therefore, been assessed by immunohistology in combination with microscopic analysis of skin sections taken from lesional as well as nonlesional vitiligo skin. A panel of antibodies specific for different properties of the melanocyte was used. Antigens associated with melanization were not found in lesional skin. In addition to antibodies to pigmentation-associated antigens, including NKI-beteb and HMB45, recognizing the pMel-17 or gp 100 protein, and MEL-5 and TMH-2 recognizing TRP-1,20-23 antibodies to various other aspects of melanocytes were also employed. The panel included NKI-C3 against a 25-100 kD differentiation antigen; G7E2 against a 110-120 kD glycoprotein; K-l2-58 recognizing an antigen downregulated during malignant pigment-cell tumor progression; 15-75 against a glycoprotein of 75 kD; Pal-Ml, which identifies the transferrin receptor and Pal-M2 against a 95-kD progression-associated glycoprotein; B9-1-12 recognizing HLA-ABC; S-100 against the calcium-binding neuropeptide of the same identity; Me114, MELl, G7A5 and AMF6, directed towards various epitopes of the highmolecular-weight protein (HMW antigen) found in malignant melanoma and associated with cell spreading; AMF7 against the alpha, integrin subunit associated with cell adhesion; and Dl-12 to MHC class II molecules.zJ-35 An important point is that this panel included antibodies reactive with melanocytes independent of their melanogenic capacity. Immunostaining of vitiligo lesional skin using polyclonal antiserum S-100 is shown in Fig 1. As this antiserum is reactive with Langerhans cells as well, a double staining with anti-
body OKT-6 to Langerhans cells was performed. Whereas in nonlesional skin single-stained melanocytes were detected in the basal layer of the epidermis, it can be observed that in lesional skin all S-100 stained cells are Langerhans cells; thus, melanocytes expressing S100 are absent from vitiligo lesional skin. This observation was confirmed for all other antibodies in the panel as well. Another important antibody to be added to the panel is reactive with the C-KIT stem cell factor receptor.36 As mentioned above, c-KIT is expressed during embryogenesis as melanocytes migrate from the site of the neural crest to their ultimate destination within the skin.37 Consequently, C-KIT is a useful marker to reveal melanocyte precursors that may prove important in repopulating vitiligo skin.18 Within the epidermis, the melanocyte is the cell type that expresses c-KIT. Others have reported this receptor, expressed by mast cells and melanocytes, to be absent in vitiligo-lesional epidermis.39 Other aspects of melanocytes represented by specific antisera remain to be investigated concerning their expression in vitiligo skin, including the endothelin-1 and FGF receptors .40,41To date, all immunohistologic evidence has appeared to support the absence of melanocytes from vitiligo lesions. Skin spanning the borders of progressive vitiligo lesions contains melanocytes of severely altered morphology.-12 Fragmented melanocytes can be observed by confocal laser scanning microscopy, and melanocytes are either grossly enlarged or display retracted dendrites. This is represented in Fig 2. These data are suggestive of premature melanocyte death. As melanocytes are lost from vitiligo lesions, effective treatment modalities must include means to repopulate lesional skin by melanocytes. This can be achieved by surgical means; transplantation of autologous melanocytes has proven effective in many cases of stable vitiligo.4”-46 Alternatively, perilesional melanocytes can potentially be stimulated to migrate and proliferate by using factors that appear to stimulate these processes in melanocytes grown in monoculture. Replacement of such stimulatory factors can possibly decelerate loss of melanocytes in progressive vitiligo as we11,d7 however, the ultimate cause of melanocyte loss may remain, leading to newly arising lesions. It is therefore of major importance to understand all factors contributing to loss of melanocytes in vitiligo.
Vitiligo
Melanocytes
Are Intrinsically
Aberrant
The morphology, proliferation rates, longevity, and melanogenic activity of melanocytes appears to vary according to the applied culture conditions.2 It has been described that melanocytes require two types of mitogenie factors for optimal propagation. The first either activates a tyrosine kinase or induces the phosphoinositol pathway, and the second stimulates the signal
Figure
1.
Frozen section cf lesio?lal skin from a vitiligo
OKT-6 in b/w. Ctdk expwssi~l,~ SIOO xcre all OKT6’,
patient immune double stained for SlOO in red and Langerham cell tr&w confirming the absence of SIOO-positive melanocytes xGfkm tkc; !eGorr. f&71,
51)
kill
transduction pathway that is dependent on c-AMPprotein kinase A. 4x-5oUnder optimal culture conditions, melanocytes are easily cultured from control as well as nonaffected vitiligo skin; 51,52however, distinctive differences in the physiology of vitiligo melanocytes were observed when compared with control counterparts in suboptimal growth medium. Vitiligo melanocytes displayed a lag period in the onset of proliferation, and they could not be subcultured as efficiently as control melanocytes.s3 At the subcellular level, these aberranties manifested as dilated rough profiles of endoplasmic reticulum (ER) and aberrant melanosome clustering in melanocytes cultured from a majority of vitiligo patients, as revealed by electron microscopy.5”,55 It has been hypothesized, that ER dilation may be a consequence of retention of TRP-1 protein in the ER.56 As mentioned, overt differences in cellular morphology were observed, particularly when cells are maintained under limiting culture conditions; thus, vitiligo melanocytes are relatively sensitive to environmental conditions. Results obtained using the fowl model for vitiligo are supportive of such differential sensitivity.s7 These data are also in accordance with preliminary in vivo observations supporting melanocyte apoptosis in vitiligo.58 In spite of the fact that mortality of vitiligo
melanocytes may depend on a plethora of different factors, such preliminary data provide an important contribution to understanding the process of the disappearance of melanocytes in vitiligo. Melanocytes themselves generate radical species in the course of melanogenesis. The orthoquinones that are generated as highly reactive intermediate metabolites can themselves contribute to the generation of a hostile environment in particular when leakage occurs from melanosomes into the cytosoL-5” In fact, substances generated in the course of melanogenesis are potentially genotoxic; it was observed that melanin yields intermediate substances that can bind to DNA.“” Although the pathway into the nucleus is still obscure, and DNA binding is not equal to causing mutations, affinity for DNA will certainly contribute to mutagenicity. Eventual benign mutations can ca~.~sevulnerability of melanocytes to further damage in \+iligo or, in a concurrent scenario, mutations ma)’ lead to melanoma by malignant transformation. If indeed vitiligo melanocytes are intrinsicalfy aberrant, this should translate into mutations in genes pivotal to melanocyte maintenance. Although involvement of hereditary factors has long been hypothesized,“‘,h2 the nature of genes affected in vitiliq> has not been
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Figure 2. Confocal laser scannirzg images of NKI-beteb imrnulostaitl led split skin preparations of (A) nonlesional, (B) perilesional, and (C) lesional skin from a vitiligo patient. The lesion had remained stab1‘e for a period of 6 months prior to taking the biopsies. Bar, 50 km. (Reprinted with permission, from the Journal of lnuestigatiue Deumatc dogy, 1993.
elucidated. So far it has been shown that in the Bangalore community, which has an increased incidence of vitiligo due to intermarriage over several generations, the disease appears not to be linked to the MITF-locus as in Waardenburg syndrome type II and in the uitmouse model for vitiligo .63,@The involvement of genetic factors is much more apparent for congenital pigmentary disorders such as piebaldism or albinism.65 The underlying mutations for many such pigmentary
disorders have already been clearly defined; however, in acquired pigmentary disorders, the role of eventual mutations that accompany changes in skin color remains obscure.66In fact, acquired depigmentation in the general population is frequently a consequence of infectious or inflammatory conditions.b7 It is intriguing that involvement of viral infections has also been proposed for vitiligo; 68 however, in nonvitiliginous individuals, depigmentation does not expand beyond the
inflicted site and is mostly reversible within a reasonable time span. In contrast, within the vitiligo patient a ‘domino effect’ can occur, generating an expanding milky-white lesion. This appears to support the notion that vitiligo-melanocyte physiology differs fundamentally from that of its unaffected counterpart. The search for genetic mutations underlying the etiology of vitiligo is possibly complicated by the fact that epidemiologic studies indicate multilocus involvement in vitiligo,69,70 and that aberrant gene expression may influence melanocyte function only under limiting circumstances. It is therefore of great interest to define environmental conditions contributing to melanocyte loss in vitiligo.
The Milieu May of Melanocytes
Contribute
to Loss
As mentioned above, the generation of radical species within the skin by melanocytes or by inflammatory cells may contribute to loss of melanocytes in vitiligo. A similar process is thought to occur during loss of pigmentation in occupational vitiligo.71,72 Although the presence or absence of melanocytes following treatment with bleaching phenolic agents has not been thoroughly investigated, the histologic appearance of skin following treatment is similar to that in generalized vitiligo.73 The persistence and spreading of depigmentation seem to suggest that melanocytes disappear from the lesions, in generalized vitiligo. The phenolic as is observed compounds are thus selectively cytotoxic for melanocytes within the skin. In monoculture, melanocytes vacuolize, retract their dendrites, detach, and die following treatment with 4 tertiary butyl phenol (4TBP), as illustrated in Fig 3.74 For components such as hydroquinone, 4-hydroxyanisole, N-acetyl-4-S-cysteaminylphenol, and alpha-methyl-4-S-cysteaminylphenol, cytotoxicity appears to be based upon such components serving as tyrosinase substrates, amounting to selective melanocytotoxicity.75-i7 Whether tyrosinase action explains selectilre cytntoxicity to melanocytes in vivo remains a matter of debate. With respect to consequences of exposure to phenolic and catecholic agents, it is of interest that several detoxifying enzymes possess limiting activity in vitiligo skin.:” -RI Several melanocyte functions may be compromised under these conditions, among them melanocyte adhesion. This may explain loss of melanocytes, in spite of the fact that no intrinsic differences in adhesive capacity were observed between control and vitiligo melanocytes?’ Because melanocytes are neural-crest-derived cells, it follows that melanocytes may respond to neurologic stimuli. This is supported by microscopic observations that demonstrate direct contact between melanocytes and nerve fibers, suggesting innervation of melano-
involvement in I-itiligo has cytes. l7 In fact, neurologic often been proposed for vitiligo? on melLtnncyte survival Environmental influences are also determined by neighboring keratinocytes. In this respect, the observation that keratinocytes Jemonstrate morphologic aberrations extending outside the circumference of the lesions indicates that .kera tinocytes may contribute to loss of melanocytes in vitiligo.sJ The intimate positioning of the 2 cell type% is suggestive of mutual interaction. Physical contact bettveen melanocytes and keratinocytes is needed to su+tnin rnelanocvtc
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growth and differentiation in keratinocyte medium;ss in addition, keratinocytes provide the source for a number of survival factors crucial for melanocyte maintenance such as bFGF and endothelin.86-s9 Among these, bFGF appears to reverse growth defects noted in melanocyte cultures derived from vitiligo patients.9” Preliminary results obtained in cocultures were suggestive of keratinocyte involvement in vitiligo;91 however, these findings have not been confirmed in a skin equivalent mode1.92Overall, the role of keratinocytes in vitiligo etiology has never been fully clarified. Stress resulting from the conditions discussed above can contribute to vitiligo etiology by altering the antigenie profile of the melanocyte. This condition may subsequently elicit an autoimmune response, as discussed below.
Cell-Mediated
Autoimmunity
Altered antigenic profiles of stressed vitiligo melanocytes may evoke an immune response in which the prevalence of humoral or cellular immunity is determined both by the cellular alterations involved and by the genotyp’e of the donor. In this respect, it is of interest that previous investigations have associated vitiligo with possession of specific subsets of MHC molecules as compared to the general population.93-95 Because the process of depigmentation takes place in perilesional skin, this is the preferential site for analysis of immunologic events associated with depigmentation. When biopsies are taken to span the lesional border and meticulously oriented to include both lesional and nonlesional skin in thin sections, minute cellular infiltrates were consistently observed in progressive lesional borders from cases of inflammatory as well as from generalized vitiligo. 96-98The fact that vitiligo is not associated with major inflammatory infiltrates is best explained by the fact that melanocytes are sparsely distributed throughout the skin. Therefore, a minute infiltrate will suffice to kill and clean up the target cells. For a detailed characterization of such infiltrates, further analysis has been performed using biopsies obtained from patients with rare cases of inflammatory vitiligo.99 In these patients, the lesion is surrounded by an inflamed, sometimes elevated border that can be itchy and appears reddish-brown. Infiltrates are overtly present in expanding lesions from these patients, and thus these infiltrates can be readily examined. It was found that these borders contain an increased number of T cells, as depicted in Fig 4. Infiltrating T cells are predominantly of the CD68+ cytotoxic subtype. Macrophages of the CD36- subtype were also found in increased numbers within the inflammatory borders. No B cells were observed in perilesional inflammatory vitiligo skin, although the presence of B cells has been reported in skin infiltrates from a patient presenting
Figure 4. Preseme of T cells in dermal irlfiltrntes nmi nlorzg the basal nzenzbrane in perilesioml skirr of a pntieut with progressive gezeralized vitiligo. Anti-CD3 immunostaining offrozen sections obtained from (A) non-lesional, (B) perilesionnl, and (C) lesional skin of a patient with inf7ammafory vitiligo. Melanin is abundant in ceils in the basal layer of nonlesional skin. Bar, 100 km.
Vogt-Koyanagi-Harada syndrome with associated vitiligo.loo Natural killer cells do not appear to be involved in melanocyte destruction in viti1igo.r”’ By interleukin-2 (IL-2) receptor immunostaining, perilesional T cells were found to be activated. The location of T cells, lining the basal layer of the epidermis and juxtapositionally opposed to remaining melano-
cytes, is very suggestive of a role for cell-mediated immunity in delivering the ‘final hit’ in melanocyte disappearance, possibly followed by clearance of cell debris by macrophages. Expression of MHC class II molecules by a subpopulation of perilesional melanocytes (Fig 5) is also in support of the role T cells appear to have in melanocyte destruction.*02 The in situ findings discussed above are in agreement with experimental data demonstrating that melanocytes are immunocompetent cells. In vitro studies have shown that melanocytes can express markers indicatik-e of immunological functions;‘03 moreover, melanocytes are capable of phagocytosis, and they can process and present mycobacterial antigens to CD4+ proliferative as well as cytotoxic T cells.‘04Jo” Melanocytes can produce a number of immunologically important cytokines, including tumor necrosis factor-alpha (TNF-tr), IL-l, IL-?, IL-G, and IL-8;1”“,10h,107 moreover, absence of melanocytes in vitiligo lesional skin coincides with decreased effectivity of Langerhans cells, which is suggestive of a role for melanocytes in Langerhans-cell function. Iox The fact that cellular infiltrates are present in vitiligo skin simultaneous with melanocyte disappearance is very suggestive of a causative role for cell-mediated
immunity in melanocyte destruction. The imbalance in peripheral T cells reported in vitiligo may ha\,e significance in this regard; l”‘l,lLo however, ultimate proof for a causative role has yet to be provided. The fact that less overt immune infilt-rates of comparable composition are also found in patients with progressive generalized vitiligo supports the notion that participation of cell-mediated immunity is a concept that applies to a much more widespread group of vitiligo patients than was previously believed. Immunodominant antigens in re,lctivity to malignantly transformed melanocytes appear to be predominantly differentiation rather then progression antigens, ‘I1 mil? among them MART-l: Pmt>Ll7, TRP-1, and tyrosinase.1’A-“7 Thus, immune reactilzitv to melanoma cells is directed against antigens shartad by melanoma cells and melanocytes. IP-Ix It is theretore conceivable that immunodominant antigens ‘ire similar in melanoma and vitiligo. The occurrence irf Cl halo ncvus phenomenon appears to support recognition of similar antigens, l2 i as does the occurrence ot’ vitiligo-like lesions in melanoma patients undergorng remission of their tumors.“’ It follows that \Gtiligo patients may be protected from melanoma, whereas mralanoma patients may be predisposed for vitiligo;lzz rm~reovt’r. immune
870
LE POOLE
AND
DAS
reactivity to vitiligo melanocytes may thus be regarded as the effective variant of an immune response that is often ineffective in melanoma. The humoral response described for vitiligo patients, particularly in the active phase of their disease, may be an important secondary phenomenon contributing to the fact that autoimmunity is effective in vitiligo, whereas in melanoma it is not.i’a-126 It follows that if means are found to interfere with immune reactivity to melanocytes in vitiligo, further depigmentation may be prevented. Further analysis of local immune reactivity and the identification of common and differing target antigens may lead to the development of new therapeutic modalities for vitiligo as well as for melanoma.rz7
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ccptor and is a progression marker in melanocytic lesions. J Invest Dermatol 1990;95:65-9. Ruiter DJ, Dingjan GM, Steylen PM, et al. Monoclonal antibodies selected to discriminate between malignant melanomas and nevocellular nevi. J Invest Dermatol 1985;85:4-8. Malissen B, Rebai N, Liabeuf A, et al. Human cytotoxic T-cell structure associated with expression of cytolysis. I. Analysis at the clonal level of the cytolysis inhibitory effect of seven monoclonal antibodies. Eur J Immunol 1982;12:73Y -47. Gaynor R, lric R, Morton D, et al. SlOO protein is present in cultured human malignant melanomas. Nature 3980; 286:400-l. Carrel S, Accola RS, Carmagnola AL, et al. Common human melanoma-associated antigens detected by monoclonal antibodies. Cancer Res 1980;40:2523-8. Puke1 CS, Lloyd KO, Travassos LS, et al. GD3, a prominent ganglioside of human melanoma. J Exp Med 1982; 155: 1133-17. de Vries JE, Keizer GD, te Velde AA, et al. Characterization of melanoma-associated surface antigens involved in the adhesion and motility of human melanoma cells. Int J Cancer 1986;38:465-73. Carrel S, Tosi R, Gross N, et al. Subsets of human Ia-like molecules defined bv monoclonal antibodies. Mol Immunol lY81;18:403-11. Gadd SJ, hshman LK. A murine monoclonal antibody specific for a cell surface antigen expressed by a subgroup of human myeloid leukemias. Leuk Res 1985;9: 1329-X. Wehrle-Hailer B, Weston JA. Soluble and cell-bound forms of steel factor activity play distinct roles in melanncvte precursor dispersal and survival on the neural crest migration pathway. Development 1995;121:731-42. Grichnik JM, Ali WN, Burch JA, et al. KIT expression reveals a population of precursor melanocytes in human skin. J Invest Dermatol 1996;106:967-71. Dippel E, Haas N, Grabbe J, et al. Expression of the c-kit receptor in hypomelanosis: a comparative study between piebald&, naevus depigmentosus and vitiligo. Br J Dermatoi 1995;132:182-9. Shannon TR, Hale CC. Identification of a 65 kDa endothelin receptor in bovine cardiac sarcolemmal vesicles. Eur J Pharmacol 1994;267:233-8. Halaban R. Growth factors and tyrosine protein phosphatascs in normal and malignant melanocytes. Cancer Metastasis Rev 1991;10:129-40. lyengar B, Misra RS. Neural differentiation of melano<$tes in vitiliginous skin. Acta Anat 1988;133:62-5. Kchl RN. ‘Treatment of vitiligo with homologous thin Thiersch’s skin grafts. Curr Med Pratt 1964;8:218-21. Falabclla R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649-55. Falabella F, Escobar C, Borrero I. Treatment of refractory ‘tnd stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26:230-6. Olsson MJ, Moellmann G, Lerner AB, et al. Vitiligo: Repigmentation with cultured melanocytes after cryostorage. Acta Derm Venereol 1994;74:226-8.
47. Morelli JG, Kincannon J, Yohn JJ, t’~ ni. Influence of inflammatory mediators and cytokines on human melanocyte function. J Invest Dermatol 1992;98:29t)d. 48. Abdel-Malek ZA, Swope VB, Nordlund JJ. Phr nature and biological effects of factor> rcsponsihle for proliferation and differentiation of melanoc!?e~ Pigment Cell Res 1992;52:43-7. 49. Swope VB, Medrano EE, Smalara 1.X cc #II. 1.ong-term proliferation of human melanocytes IS supported by the physiologic mitogens alpha-melanotropin, endothelin-1, and basic fibroblast growth factor 1.xy: Cell lies I995:2 17: 453-9. 50. Smit NPM, Westerhof W, Menko W, e: al. Stimulation 01 cultured melanocytes in medium containing ‘3 serum substitute: Ultroser-G. Pigment Ceil Rcbs 1995;8:79--27’. 51. Medrano EE, Nordlund JJ, Succesfu! culture of adult human melanocytes from norm<11 ;~nd \-itiligo donors. 1 Invest Dermatol 7 990;95:443 -5. 52. Im S, Harm SK, Park YK, et aJ. Culture of mclanocvtes obtained from normal and vitiligo s;l~h!ect<. Yonsei Med [ 1992;33:344-50. 53. Puri N, Mojamdar M, Ramaiah A. In i rtro growth characteristics of melanocytes obtained from adult normal and vitiligo subjects. J Invest DermattJl l987;88:434-8. 54. Im S, Harm SK, Kim HI, et al. BitQ$ characteristics of cultured human vitiligo melanocvte~ int 1 Dermatol 1994,33:556-62. ,.* <55. Boissy RE, Liu Y, Medrano EE, et a!. Qructur~tl aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-t
61. Merelender J, Rywlin J. A propos de !‘lGrc?ditr du vitiligo acyuis (vitiligo dans 3 generations). .41 tn Derm Venereal 1940;121:583-5. 62. Majumder I’, Nordlund JJ, Nat11 S i’attern i>f familial aggregation of vitiligo. Arch J Dermaeoi 1993;~129:994-8. 63. Ramaiah A, Mojamdar MV, Amarnath VM. Vitiligo in the SSK community of Bangalor. indI,m 1 Dcrmntol Vc-nereol Lepr 1988:54:2X-4. 64. Tripathi RK, Flanders DJ, Young ‘TX., %.t,11.E\ aluation of MIFF locus linkage to human vitiligcl and osteopetrosis (abstract). Pigment Cell Res 199h;S5:% 65. Orlow SJ. Congenital disorders of ilvl,opiGmentation. Semin Dermatol 1995;14:27-32. 66. Boissv RE, Nordlund JJ. Molccrrlar i?a
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influence of keratinocytes. Arch Dermatol Res 1993;285: 385-92. Gordon PR, Mansur CP, Gilchrest BA. Regulation of human melanocyte growth, dendricity and melanization by keratinocyte derived factors. J Invest Dermatol 1989; 92:565-72. Halaban R, Langdon R, Birchall N, et al. Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol 1988; 107:1611-9. Yohn JJ, Morelli JG, Walchak SJ, et al. Cultured human keratinocytes synthesize and secrete endothelin-1. J Invest Dermatol 1993;100:23-6. Hara M, Yaar M, Gilchrest BA. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity. J Invest Dermatol 1995;105:744-8. Puri N, Majamdar M, Ramaiah A. Growth defects of melanocytes in culture from vitiligo subjects can be partially corrected by the addition of fibroblast derived growth factor in vitro. Arch Dermatol Res 1989;281:17884. Le Poole IC, van den Wijngaard RMJGJ, Das PK, et al. Epidermal cell cocultures from healthy and vitiligo donors to study the etiopathogenesis of vitiligo. 39th Annual European Tissue Culture Society meeting 1991 (abstract). Bessou S, Gauthier Y, Pain C, et al. In vivo and ex vivo melanocyte transplants in vitiligo: An extrinsic factor is needed to trigger the disease (abstract). Pigment Cell Res 1996;S5:26. Al-Fouzan A, al-Arbash M, Fouad F, et al. Study of HLA class I/II and T lymphocyte subsets in Kuwaiti vitiligo patients. Eur J Immunogenet 1995;22:209-13. Venneker GT, de Waal LP, Westerhof W, et al. HLA associations in vitiligo patients in the Dutch population. Disease markers. 1993;11:187-90. Orecchia G, Perfetti L, Malagoli P, et al. Vitiligo is associated with a significant increase in HLA-A30, CW6 and DQw3 and a decrease in C4AQO in northern Italian patients. Dermatology 1992;185:123-7. Michaelsson G. Vitiligo with raised borders: Report of two cases. Acta Dermatol Venereol 1968;48:158-61. Ahn SK, Choi EH, Lee SH, et al. Immunohistochemical studies from vitiligo: Comparison between active and inactive lesions. Yonsei Med J 1994;35:404-10. Abdel-Nasr MB, Kruger-Krasagakes S, Krasagakis K, et al. Further evidence for involvement of both cell mediated and humoral immunity in generalized vitiligo. Pigment Cell Res 1994;1:1-8. Le Poole IC, van den Wijngaard RMJGJ, Westerhof W, et al. Presence of T cells and macrophages in inflammatory vitiligo skin parallels melanocyte disappearance. Am J Path01 1996;148:1219-28. Okada T, Sakamoto T, Ishibashi T, et al. Vitiligo in VogtKoyanagi-Harada disease: Immunohistological analysis of inflammatory site. Graefes Arch Clin Exp Ophtalmol 1996;234:359 - 63. Durham-Pierre DG, Waltters CS, Halder RM, et al. Natural killer cell and lymphokine-activated killer cell activity against melanocytes in vitiligo. J Am Acad Dermatol 1995;33:26-30. Al Badri AM, Foulis AK, Todd PM, et al. Abnormal
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expression of MHC class 11 and ICAMby melanocytes in vitiligo. J Path01 1993;169:203-6. Smit NPM, Le Poole IC, van den Wijngaard RMJGJ, et al. Expression of different immunological markers by culhwed human melanocytes. Arch Dermatol Res 1993;285: 3ih-65. Lta Poole IC, van den Wijngaard RMJGJ, Westerhof W, et al. I’hagocytosis by normal human melanocytes in vitro. Exp Cell Res 1993;205:388-95. Le Poole IC, Mutis P, van den Wijngaard RMJGJ, et al. A novel, antigen presenting function of melanocytes and its possible relation to hypopigmentary disorders. J Immunol l993;151:7284-92. Swore VB, Sauder DN, McKenzie RC, et al. Synthesis of interleukin l-alpha and beta by normal human melanocytes. J Invest Dermatol 1994;102:749-53. iachariae COC, Thestrap-Pedersen K, Matsushima K. Expression and secretion of leukocyte chemotactic cytokines bv normal human melanocytes and melanoma cc&. 1 Invest Dermatol 1991;9:593-9. C;iihar A, A&n E, @hand N, Etzioni A. Vitiliginous \ er>us pigmented skin response to intradermal administration ot interferon gamma. Arch Dermatol 1993;129: 600 - 1. flann SK, Park YK, Chung KY, et al. Peripheral blood lvmphocyte imbalance in Koreans with active vitiligo. Int J Dermatol 1993;32:286-9. Abdel-Naser MB, Ludwig WD, Gollnick H, et al. Nonsegmental vitiligo: Decrease of the CD45RAt- T-cell subset and ebidencr for peripheral T-cell activation. Int J I>erm,ltol 1992;31:321-6. %arour f-I, De Smet C, Lehrmann F, et al. The majority of autologuu\ cytolytic T-lymphocyte clones derived from peripheral blood lymphocytes of a melanoma patient rccogni/.e an antigenic peptide derived from gene I’mell7/gplOR J Invest Dermatol 1996;107:63-7. Houghton AX Cancer antigens: Immune recognition of >elf and altered self. J Exp Med 1994;180:1-4. Anichini A, Maccalli C, Motarini R, et al. Melanoma cells ,md normal melanocytes share antigens recognized by IHLA-AZ restricted cytotoxic T-cell clones from melanctma patients. J Esp Med 1993;177:989-98. Cvulie PC,, Brichard V, van Pel A, et al. A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Eup Med 1994;180:35-42. Bakker AB, Schreurs MW, de Boer AJ, et al. Melanocyte lineage-specific antigen gplO0 is recognized by melano-
ma-derived tumor-infiltrating lympht+~tc~ J Fxp Med 1994;179:1005-9. 116. Hara I, Take&i Y, Houghton AN. Implicating a role for immune recognition of self in tumor rejection: Passive immunization against the brown locas protein, I Exp Med 1995;182:1609-14. 117. Brichard V, van Pel A, Wiilfel ‘I, ct .\I. .Ihe tVrosinasc gene codes for an antigen rccognizcd by rr~tologous cytolytic T lymphocytes on t-il./\-Al ^nc~lanorna~. 1 Exp Med
1993;178:489
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128. Parmiani C. Tumor immunity ah autt~immrmit~: Tumot antigens include normal self profeinc t\,hich stimulate anergic peripheral T cells. lmmunol T~tti+ I W3;14:536-8. 119. Cui J, Bystryn JC. Melanoma and \-itilrgc.1 are associated with antibody responses to similar aniigc>nh c:n pigment cells. Arch Dermatol 1995;131 :?I -i-- & 120. Rosenberg SA, White DE. Vitiligo in vat&t-s with melanoma: Normal tissue antigens can bc? targets tar cancer immunotherapy. J Immunother 19%;: Lj:dl-.+ 121. Tokura I’, Yamanaka K, Watika 11, rt ,,I. Halo congenital nevus undergoing spontaneous re;;rc+.+ln: Involvement of T-cell immunit!, and presenct> r>t ( .rculating anti-nrTIE it>11 IgM antibodies. ,\rch l>~rm~>:oi lY~~-1;‘130:101641. 122. Scheidenbogen C, Hunstcin VV, l\;c~~ih!11%U. \‘itiligo-like> lesions following immunotherap\. w:;h 1F% alpha dnd IL-2 in melanoma patients Eur ] ( iini “r !9%:iOA:l2(19-Il. 123. Cui J, Harning M, Hcnn M, et i-11 iclt!ntitlcatit,n 01 pigment cell antigens bv vitiligo antlhocl~~.‘~ 1 In\ cst Drnnato1 19Y2;98:162-3. 124. Haming R, Cui J, Bvstryn JC. lit>latioir between the incidence and level of .pigment ccl1 and d&nsc activity in vitiligo. J Invest Derm&ol 199 i;%‘: 1013 -- 80. 125. Norris DA, Kissinger RM, Naughton <.;M, et al. Evidence for immunologic mechanisms in human vitiligo: Patients’ sera induce damage to hum,:n melanocyte5 in \,itro by complement-mediated dam3gc~ and antibodydependent cellular cytotouicit\ I Ini ikst l)c?rnratol 1988: 90:783-c). 126. Gilhar A, Zelicksnn B, Ulman ‘I’.
Microscopic
Changes in Vitiligo
I. CAROLINE LE POOLE, PRANAB K. DAS, PhD
V
PhD
itiligo is characterized by acquired depigmentation in expanding lesions of the skin.] Microscopic ’ analysis has contributed greatly to our present level of understanding of the etiopathology of vitiligo. Light microscopy has been used to reveal changes in the skin associated with depigmentation at the cellular level. Electron microscopy has multiplied the level of magnification roughly by a factor of 100, useful to examine more intimate details at the subcellular level. Combining microscopy with the use of labeled antibodies, the resolution of microscopy has been raised to the molecular level, so that the presence or absence of specific antigens can easily be followed under a variety of circumstances. These methods have provided a means to examine the physiology of melanocytes in conjunction with various components of the tissue environment. Microscopic analysis is of particular use for enigmatic diseasessuch as vitiligo, where a comparison of diseased versus healthy tissue can further direct research concerning the pathomechanism of the disease. Results from these studies have implemented functional assays that use various model systems, including monocultures of melanocytes, cocultures, skinequivalent models, organotypic culture of human skin explants, and animal models.2-’ The development of effective treatment is dependent on an understanding of the events leading to depigmentation in vitiligo. This marks the importance of investigations undertaken to determine whether dormant melanocytes remain within the depigmented lesions, or whether loss of pigmentation involves loss of pigment ceils altogether. Using in vitro cultured cells, the physiology of melanocytes from control and from vitiligo donors has been compared within a defined and adjustable environment. Using cultured cells, evidence has been acquired for potential intrinsic abberancies of vitiligo melanocytes. It must be borne in mind however, that depigmentation in vivo may ultimately be brought
about by environmental factors. In this respect, surrounding keratinocytes may generate a hostile environment for melanocytes. Within a hostile environment, the pigment cell is particularly vulnerable because of its low turnover rate. The histology of perilesional as well as nonlesional vitiligo skin can be informative as to the physiology of melanocytes as well as to cells and matrix molecules comprising the direct environment of the pigment cell. It is conceivable that, at the clinical level, melanocytes appear to be selectively affected, whereas in actual fact, the environment is altered as well. Immunohistochemical evidence is accumulating to show that cellular immunity is involved in vitiligo. In addition to humoral immunity as discussed in the previous chapter, T cells and macrophages are potentially involved in the process of depigmentation. The mode of pigment elimination proposed here would suggest vitiligo to be the effective variant of an in situ immune reaction that is active, but ineffective, in melanoma. The outcome of in viva and in vitro studies appears to support the view that vitiligo is
Loss of Melanocytes
in Vitiligo
Skin
Depigmentation of the skin is potentially a consequence of either of two conditions: (1.) melanocytes may be absent from depigmented skin, or (2) melanogenesis may have been silenced in melanocvtes still present within the lesion. Examples of the absence of melanocytes in depigmented lesions exist in piebaldism, where melanocytes do not populate all areas of the skin during development as a consequence of mutations in the (.-KIT stem cell factor receptor and in Waardenburg syndrome types I-III, where focal absence of melanoeytes appears to be a consequence of mutations in the PAX or MITF transcription factor genes.y 1’) In contrast. silenced mel-
anogenesis is found in albinism, where pigmentation either is reduced or is totally absent as a consequence of mutations in pigmentation-associated genes, to an extent dependent on the type of mutation as well as on the pigmentary gene affected. 11-13 Reduced pigmentation indicates that melanocytes are still present, as in oculocutaneous albinism (OCA) types II and III. It follows that when pigmentation is totally absent from the lesions as in vitiligo, the presence or absence of melanocytes remains an open question. Occasionally, melanocyte cultures can be initiated from lesional vitiligo skin (personal observation); moreover, it has been reported that residual tyrosinase activity remains in vitiligo lesions. 14 As activity of this enzyme is solely restricted to melanocytes, it appears that at least a subpopulation of melanocytes remains present within the lesion. Dormant melanocytes can potentially be detected by histological analysis at the light microscopic (LM) or electron microscopic (EM) level.r5J6 It has been reported that neither histology nor electron microscopic investigation of lesional skin revealed the presence of any ‘clear cells’ within the lesion;lT-I9 however, it can be argued that certain melanocytic properties change along with the observed loss of melanization. Presence or absence of melanocytes in vitiligo lesions has, therefore, been assessed by immunohistology in combination with microscopic analysis of skin sections taken from lesional as well as nonlesional vitiligo skin. A panel of antibodies specific for different properties of the melanocyte was used. Antigens associated with melanization were not found in lesional skin. In addition to antibodies to pigmentation-associated antigens, including NKI-beteb and HMB45, recognizing the pMel-17 or gp 100 protein, and MEL-5 and TMH-2 recognizing TRP-1,20-23 antibodies to various other aspects of melanocytes were also employed. The panel included NKI-C3 against a 25-100 kD differentiation antigen; G7E2 against a 110-120 kD glycoprotein; K-l2-58 recognizing an antigen downregulated during malignant pigment-cell tumor progression; 15-75 against a glycoprotein of 75 kD; Pal-Ml, which identifies the transferrin receptor and Pal-M2 against a 95-kD progression-associated glycoprotein; B9-1-12 recognizing HLA-ABC; S-100 against the calcium-binding neuropeptide of the same identity; Me114, MELl, G7A5 and AMF6, directed towards various epitopes of the highmolecular-weight protein (HMW antigen) found in malignant melanoma and associated with cell spreading; AMF7 against the alpha, integrin subunit associated with cell adhesion; and Dl-12 to MHC class II molecules.zJ-35 An important point is that this panel included antibodies reactive with melanocytes independent of their melanogenic capacity. Immunostaining of vitiligo lesional skin using polyclonal antiserum S-100 is shown in Fig 1. As this antiserum is reactive with Langerhans cells as well, a double staining with anti-
body OKT-6 to Langerhans cells was performed. Whereas in nonlesional skin single-stained melanocytes were detected in the basal layer of the epidermis, it can be observed that in lesional skin all S-100 stained cells are Langerhans cells; thus, melanocytes expressing S100 are absent from vitiligo lesional skin. This observation was confirmed for all other antibodies in the panel as well. Another important antibody to be added to the panel is reactive with the C-KIT stem cell factor receptor.36 As mentioned above, c-KIT is expressed during embryogenesis as melanocytes migrate from the site of the neural crest to their ultimate destination within the skin.37 Consequently, C-KIT is a useful marker to reveal melanocyte precursors that may prove important in repopulating vitiligo skin.18 Within the epidermis, the melanocyte is the cell type that expresses c-KIT. Others have reported this receptor, expressed by mast cells and melanocytes, to be absent in vitiligo-lesional epidermis.39 Other aspects of melanocytes represented by specific antisera remain to be investigated concerning their expression in vitiligo skin, including the endothelin-1 and FGF receptors .40,41To date, all immunohistologic evidence has appeared to support the absence of melanocytes from vitiligo lesions. Skin spanning the borders of progressive vitiligo lesions contains melanocytes of severely altered morphology.-12 Fragmented melanocytes can be observed by confocal laser scanning microscopy, and melanocytes are either grossly enlarged or display retracted dendrites. This is represented in Fig 2. These data are suggestive of premature melanocyte death. As melanocytes are lost from vitiligo lesions, effective treatment modalities must include means to repopulate lesional skin by melanocytes. This can be achieved by surgical means; transplantation of autologous melanocytes has proven effective in many cases of stable vitiligo.4”-46 Alternatively, perilesional melanocytes can potentially be stimulated to migrate and proliferate by using factors that appear to stimulate these processes in melanocytes grown in monoculture. Replacement of such stimulatory factors can possibly decelerate loss of melanocytes in progressive vitiligo as we11,d7 however, the ultimate cause of melanocyte loss may remain, leading to newly arising lesions. It is therefore of major importance to understand all factors contributing to loss of melanocytes in vitiligo.
Vitiligo
Melanocytes
Are Intrinsically
Aberrant
The morphology, proliferation rates, longevity, and melanogenic activity of melanocytes appears to vary according to the applied culture conditions.2 It has been described that melanocytes require two types of mitogenie factors for optimal propagation. The first either activates a tyrosine kinase or induces the phosphoinositol pathway, and the second stimulates the signal
Figure
1.
Frozen section cf lesio?lal skin from a vitiligo
OKT-6 in b/w. Ctdk expwssi~l,~ SIOO xcre all OKT6’,
patient immune double stained for SlOO in red and Langerham cell tr&w confirming the absence of SIOO-positive melanocytes xGfkm tkc; !eGorr. f&71,
51)
kill
transduction pathway that is dependent on c-AMPprotein kinase A. 4x-5oUnder optimal culture conditions, melanocytes are easily cultured from control as well as nonaffected vitiligo skin; 51,52however, distinctive differences in the physiology of vitiligo melanocytes were observed when compared with control counterparts in suboptimal growth medium. Vitiligo melanocytes displayed a lag period in the onset of proliferation, and they could not be subcultured as efficiently as control melanocytes.s3 At the subcellular level, these aberranties manifested as dilated rough profiles of endoplasmic reticulum (ER) and aberrant melanosome clustering in melanocytes cultured from a majority of vitiligo patients, as revealed by electron microscopy.5”,55 It has been hypothesized, that ER dilation may be a consequence of retention of TRP-1 protein in the ER.56 As mentioned, overt differences in cellular morphology were observed, particularly when cells are maintained under limiting culture conditions; thus, vitiligo melanocytes are relatively sensitive to environmental conditions. Results obtained using the fowl model for vitiligo are supportive of such differential sensitivity.s7 These data are also in accordance with preliminary in vivo observations supporting melanocyte apoptosis in vitiligo.58 In spite of the fact that mortality of vitiligo
melanocytes may depend on a plethora of different factors, such preliminary data provide an important contribution to understanding the process of the disappearance of melanocytes in vitiligo. Melanocytes themselves generate radical species in the course of melanogenesis. The orthoquinones that are generated as highly reactive intermediate metabolites can themselves contribute to the generation of a hostile environment in particular when leakage occurs from melanosomes into the cytosoL-5” In fact, substances generated in the course of melanogenesis are potentially genotoxic; it was observed that melanin yields intermediate substances that can bind to DNA.“” Although the pathway into the nucleus is still obscure, and DNA binding is not equal to causing mutations, affinity for DNA will certainly contribute to mutagenicity. Eventual benign mutations can ca~.~sevulnerability of melanocytes to further damage in \+iligo or, in a concurrent scenario, mutations ma)’ lead to melanoma by malignant transformation. If indeed vitiligo melanocytes are intrinsicalfy aberrant, this should translate into mutations in genes pivotal to melanocyte maintenance. Although involvement of hereditary factors has long been hypothesized,“‘,h2 the nature of genes affected in vitiliq> has not been
866
LE POOLE AND DAS
Chits
irz Dermatology
l
7997;15:863-873
Figure 2. Confocal laser scannirzg images of NKI-beteb imrnulostaitl led split skin preparations of (A) nonlesional, (B) perilesional, and (C) lesional skin from a vitiligo patient. The lesion had remained stab1‘e for a period of 6 months prior to taking the biopsies. Bar, 50 km. (Reprinted with permission, from the Journal of lnuestigatiue Deumatc dogy, 1993.
elucidated. So far it has been shown that in the Bangalore community, which has an increased incidence of vitiligo due to intermarriage over several generations, the disease appears not to be linked to the MITF-locus as in Waardenburg syndrome type II and in the uitmouse model for vitiligo .63,@The involvement of genetic factors is much more apparent for congenital pigmentary disorders such as piebaldism or albinism.65 The underlying mutations for many such pigmentary
disorders have already been clearly defined; however, in acquired pigmentary disorders, the role of eventual mutations that accompany changes in skin color remains obscure.66In fact, acquired depigmentation in the general population is frequently a consequence of infectious or inflammatory conditions.b7 It is intriguing that involvement of viral infections has also been proposed for vitiligo; 68 however, in nonvitiliginous individuals, depigmentation does not expand beyond the
inflicted site and is mostly reversible within a reasonable time span. In contrast, within the vitiligo patient a ‘domino effect’ can occur, generating an expanding milky-white lesion. This appears to support the notion that vitiligo-melanocyte physiology differs fundamentally from that of its unaffected counterpart. The search for genetic mutations underlying the etiology of vitiligo is possibly complicated by the fact that epidemiologic studies indicate multilocus involvement in vitiligo,69,70 and that aberrant gene expression may influence melanocyte function only under limiting circumstances. It is therefore of great interest to define environmental conditions contributing to melanocyte loss in vitiligo.
The Milieu May of Melanocytes
Contribute
to Loss
As mentioned above, the generation of radical species within the skin by melanocytes or by inflammatory cells may contribute to loss of melanocytes in vitiligo. A similar process is thought to occur during loss of pigmentation in occupational vitiligo.71,72 Although the presence or absence of melanocytes following treatment with bleaching phenolic agents has not been thoroughly investigated, the histologic appearance of skin following treatment is similar to that in generalized vitiligo.73 The persistence and spreading of depigmentation seem to suggest that melanocytes disappear from the lesions, in generalized vitiligo. The phenolic as is observed compounds are thus selectively cytotoxic for melanocytes within the skin. In monoculture, melanocytes vacuolize, retract their dendrites, detach, and die following treatment with 4 tertiary butyl phenol (4TBP), as illustrated in Fig 3.74 For components such as hydroquinone, 4-hydroxyanisole, N-acetyl-4-S-cysteaminylphenol, and alpha-methyl-4-S-cysteaminylphenol, cytotoxicity appears to be based upon such components serving as tyrosinase substrates, amounting to selective melanocytotoxicity.75-i7 Whether tyrosinase action explains selectilre cytntoxicity to melanocytes in vivo remains a matter of debate. With respect to consequences of exposure to phenolic and catecholic agents, it is of interest that several detoxifying enzymes possess limiting activity in vitiligo skin.:” -RI Several melanocyte functions may be compromised under these conditions, among them melanocyte adhesion. This may explain loss of melanocytes, in spite of the fact that no intrinsic differences in adhesive capacity were observed between control and vitiligo melanocytes?’ Because melanocytes are neural-crest-derived cells, it follows that melanocytes may respond to neurologic stimuli. This is supported by microscopic observations that demonstrate direct contact between melanocytes and nerve fibers, suggesting innervation of melano-
involvement in I-itiligo has cytes. l7 In fact, neurologic often been proposed for vitiligo? on melLtnncyte survival Environmental influences are also determined by neighboring keratinocytes. In this respect, the observation that keratinocytes Jemonstrate morphologic aberrations extending outside the circumference of the lesions indicates that .kera tinocytes may contribute to loss of melanocytes in vitiligo.sJ The intimate positioning of the 2 cell type% is suggestive of mutual interaction. Physical contact bettveen melanocytes and keratinocytes is needed to su+tnin rnelanocvtc
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growth and differentiation in keratinocyte medium;ss in addition, keratinocytes provide the source for a number of survival factors crucial for melanocyte maintenance such as bFGF and endothelin.86-s9 Among these, bFGF appears to reverse growth defects noted in melanocyte cultures derived from vitiligo patients.9” Preliminary results obtained in cocultures were suggestive of keratinocyte involvement in vitiligo;91 however, these findings have not been confirmed in a skin equivalent mode1.92Overall, the role of keratinocytes in vitiligo etiology has never been fully clarified. Stress resulting from the conditions discussed above can contribute to vitiligo etiology by altering the antigenie profile of the melanocyte. This condition may subsequently elicit an autoimmune response, as discussed below.
Cell-Mediated
Autoimmunity
Altered antigenic profiles of stressed vitiligo melanocytes may evoke an immune response in which the prevalence of humoral or cellular immunity is determined both by the cellular alterations involved and by the genotyp’e of the donor. In this respect, it is of interest that previous investigations have associated vitiligo with possession of specific subsets of MHC molecules as compared to the general population.93-95 Because the process of depigmentation takes place in perilesional skin, this is the preferential site for analysis of immunologic events associated with depigmentation. When biopsies are taken to span the lesional border and meticulously oriented to include both lesional and nonlesional skin in thin sections, minute cellular infiltrates were consistently observed in progressive lesional borders from cases of inflammatory as well as from generalized vitiligo. 96-98The fact that vitiligo is not associated with major inflammatory infiltrates is best explained by the fact that melanocytes are sparsely distributed throughout the skin. Therefore, a minute infiltrate will suffice to kill and clean up the target cells. For a detailed characterization of such infiltrates, further analysis has been performed using biopsies obtained from patients with rare cases of inflammatory vitiligo.99 In these patients, the lesion is surrounded by an inflamed, sometimes elevated border that can be itchy and appears reddish-brown. Infiltrates are overtly present in expanding lesions from these patients, and thus these infiltrates can be readily examined. It was found that these borders contain an increased number of T cells, as depicted in Fig 4. Infiltrating T cells are predominantly of the CD68+ cytotoxic subtype. Macrophages of the CD36- subtype were also found in increased numbers within the inflammatory borders. No B cells were observed in perilesional inflammatory vitiligo skin, although the presence of B cells has been reported in skin infiltrates from a patient presenting
Figure 4. Preseme of T cells in dermal irlfiltrntes nmi nlorzg the basal nzenzbrane in perilesioml skirr of a pntieut with progressive gezeralized vitiligo. Anti-CD3 immunostaining offrozen sections obtained from (A) non-lesional, (B) perilesionnl, and (C) lesional skin of a patient with inf7ammafory vitiligo. Melanin is abundant in ceils in the basal layer of nonlesional skin. Bar, 100 km.
Vogt-Koyanagi-Harada syndrome with associated vitiligo.loo Natural killer cells do not appear to be involved in melanocyte destruction in viti1igo.r”’ By interleukin-2 (IL-2) receptor immunostaining, perilesional T cells were found to be activated. The location of T cells, lining the basal layer of the epidermis and juxtapositionally opposed to remaining melano-
cytes, is very suggestive of a role for cell-mediated immunity in delivering the ‘final hit’ in melanocyte disappearance, possibly followed by clearance of cell debris by macrophages. Expression of MHC class II molecules by a subpopulation of perilesional melanocytes (Fig 5) is also in support of the role T cells appear to have in melanocyte destruction.*02 The in situ findings discussed above are in agreement with experimental data demonstrating that melanocytes are immunocompetent cells. In vitro studies have shown that melanocytes can express markers indicatik-e of immunological functions;‘03 moreover, melanocytes are capable of phagocytosis, and they can process and present mycobacterial antigens to CD4+ proliferative as well as cytotoxic T cells.‘04Jo” Melanocytes can produce a number of immunologically important cytokines, including tumor necrosis factor-alpha (TNF-tr), IL-l, IL-?, IL-G, and IL-8;1”“,10h,107 moreover, absence of melanocytes in vitiligo lesional skin coincides with decreased effectivity of Langerhans cells, which is suggestive of a role for melanocytes in Langerhans-cell function. Iox The fact that cellular infiltrates are present in vitiligo skin simultaneous with melanocyte disappearance is very suggestive of a causative role for cell-mediated
immunity in melanocyte destruction. The imbalance in peripheral T cells reported in vitiligo may ha\,e significance in this regard; l”‘l,lLo however, ultimate proof for a causative role has yet to be provided. The fact that less overt immune infilt-rates of comparable composition are also found in patients with progressive generalized vitiligo supports the notion that participation of cell-mediated immunity is a concept that applies to a much more widespread group of vitiligo patients than was previously believed. Immunodominant antigens in re,lctivity to malignantly transformed melanocytes appear to be predominantly differentiation rather then progression antigens, ‘I1 mil? among them MART-l: Pmt>Ll7, TRP-1, and tyrosinase.1’A-“7 Thus, immune reactilzitv to melanoma cells is directed against antigens shartad by melanoma cells and melanocytes. IP-Ix It is theretore conceivable that immunodominant antigens ‘ire similar in melanoma and vitiligo. The occurrence irf Cl halo ncvus phenomenon appears to support recognition of similar antigens, l2 i as does the occurrence ot’ vitiligo-like lesions in melanoma patients undergorng remission of their tumors.“’ It follows that \Gtiligo patients may be protected from melanoma, whereas mralanoma patients may be predisposed for vitiligo;lzz rm~reovt’r. immune
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reactivity to vitiligo melanocytes may thus be regarded as the effective variant of an immune response that is often ineffective in melanoma. The humoral response described for vitiligo patients, particularly in the active phase of their disease, may be an important secondary phenomenon contributing to the fact that autoimmunity is effective in vitiligo, whereas in melanoma it is not.i’a-126 It follows that if means are found to interfere with immune reactivity to melanocytes in vitiligo, further depigmentation may be prevented. Further analysis of local immune reactivity and the identification of common and differing target antigens may lead to the development of new therapeutic modalities for vitiligo as well as for melanoma.rz7
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ma-derived tumor-infiltrating lympht+~tc~ J Fxp Med 1994;179:1005-9. 116. Hara I, Take&i Y, Houghton AN. Implicating a role for immune recognition of self in tumor rejection: Passive immunization against the brown locas protein, I Exp Med 1995;182:1609-14. 117. Brichard V, van Pel A, Wiilfel ‘I, ct .\I. .Ihe tVrosinasc gene codes for an antigen rccognizcd by rr~tologous cytolytic T lymphocytes on t-il./\-Al ^nc~lanorna~. 1 Exp Med
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