Pathophysiology of vitiligo

Pathophysiology of vitiligo

ELSFVIER Pathophysiology of Vitiligo JEROME CASTANET, MD JEAN-PAUL ORTONNE, MD V itiligo is a common skin and hair disorder affecting approximate...

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of Vitiligo



itiligo is a common skin and hair disorder affecting approximately l-4% of the world’s population and characterized by circumscribed white spots on the skin that tend to enlarge centrifugally over time .1,2 The course of vitiligo on a case by case basis is unpredictable. The natural course of the disease is usually one of slow progression, but it may stabilize or exacerbate rapidly. Some degree of spontaneous or sun-induced repigmentation is not uncommon in vitiligo, but complete and stable repigmentation is a rare event. The characteristic histological picture is the total absence of melanin and melanin-forming cells, or melanocytes, with an otherwise normal dermis and epidermis. The etiology is unknown, and several hypotheses have been proposed to explain the loss of melanocytes. The autocytoxic hypothesis supposes that intermediate metabolites in the melanin synthesis are melanocytotoxic. The neural hypothesis suggests that accumulation of some neurochemical mediator causes decreased melanin production. The autoimmune hypothesis is the most prevalent, and it is based mainly on the presence of melanocyte-specific antibodies that are able to induce necrosis of cultured human melanocytes:’ Recent advances in melanocyte research, due largely to the availability of techniques for culturing normal human melanocytes, opened new perspectives in the understanding of vitiligo. Intrinsic melanocyte defects, genetic factors, biochemical or neurotransmitter imbalance, abnormal free-radical defense mechanisms, and abnormal responses to growth factors might be involved in the development of vitiligo.‘J As each of these hypotheses bears its own line of evidence, composite theories have been proposed, merging all theories to explain all the findings observed in vitiligo.’ The description of so many credible contributory factors to the pathogenesis of vitiligo was also recently addressed in a “convergence theory” in which different causal factors may act independently or synergystically to induce disappearance of melanocytes.2 Vitiligo might be a heterogeneous process in which multiple factors, varying in different patients and with the stages of the disease, influence the melanocyte biological behavior.

The question is raised whether vitiligo is a syndrome or a single disease.’

What is the behavior macules?

of melanocytes

in vitiligo

Ultrastructural studies have demonstrated that cells with structural characteristics of melanocytes are not present in established vitiligo skin. Histochemical studies, using extensive panels of monoclonal antibodies specific for various melanocyte antigens, did not show any staining of pigment cells, even inactive, in vitiligo epidermis.4 These findings strongly support the view that melanocytes are destroyed in established vitiligo. The characteristic histologic picture of vitiligo is an absence of melanocyte and melanin pigment. Pictures of melanocyte destruction by hemophagocytosis are very rare, if any. Although complement-fixing antibodies might be expected to produce an intense neutrophilic infiltrate, there is no such infiltrate in vitiligo skin. In vitiligo with erythematous borders, only sprinkles of mononuclear cells and lymphocytes are present at the edge of the lesion. The histologic picture does not argue for a major role of autoimmunity during vitiligo; however, morphologic studies have emphasized subtle changes observed in the normal appearing skin adjacent to vitiliginous skin: degenerative changes in melanocytes, vacuolar changes of basal cellsF7 mild vesiculation in the epidermisH and intradermal and epidermal infiltration of lymphocytes.-Li~~7,9 These changes seem more prominent in the skin of actively spreading vitiligo than in stable vitiligo.”



There are numerous animal models that show spontaneous vitiligo-like depigmentation.

The Smyth chicken model of depigmentation is considered to be a disorder with two interacting components: melanocyte dysfunction and autoimmune reactions. Affected birds from this line express a postnatal loss of melanocytes in feather and ocular tissues. Histologic examination of the Smyth chicken skin shows a dense mononuclear infiltrate in white feather follicles that is not present in normally pigmented follicles. Results of cross-breeding studies emphasize the importance of genetic factors. The cells die as a consequence of defects in




Clinics in Drmntolog~


tyrosinase production and translocation and the engorgement of cytoplasm with autophagosomes. Melanocyte death elicits an antibody-mediated autoimmune reaction that accelerates or enhances melanocyte destruction. Antimelanocyte autoantibodies are detected in the sera of affected chicks several weeks before the onset of depigmentation. The identified auto-antibodies bind to multiple chick melanocyte proteins of between 65 and 80 kD and recognize mammalian tyrosinaserelated protein-l .*O Bursectomy, which impairs antibody responses, and cyclosporine A, which selectively inhibits T-cell-mediated immune responses, both delay the onset and reduce the severity of vitiligo, suggesting that both types of immune responses are involved in the pathogenesis of Smyth chicken depigmentation.ll-l3

The C57BL/Gj-miVit/miVit


In contrast, in the C57BL/6j-mi”it/mi”it

mouse model, the depigmentation process is not related to the immune system. 14When the pigmented skin of this mouse is grafted onto nude mice, it becomes white, showing that an intrinsic melanocyte defect rather than extrinsic factors are involved in the depigmenting process. The mouse also has a slowly progressing retinal degeneration, in which photoreceptor cell nuclei and rhodopsin are gradually lost and the retinal pigment epithelium is unevenly pigmented. It has been shown that phagosomes are significantly reduced as compared to controls.15 In these mice, an accumulation of retinyl palmitate and all-truns-retinol occurred in eyes and an alteration of systemic retinoid metabolism was demonstrated under normal dietary conditions;l6 however, it is not known whether a defect in retinal pigment epithelium or abnormalities of retinoid metabolism in eyes and liver are the direct cause of the retinal defect. The C57BL/6j-mi”it/mi”it mouse model also exhibits an immune deficiency. These animals are unable to mount a normal immune/inflammatory response upon epicutaneous application of potent contact allergens. A deficient interaction between epidermal cells and T lymphocytes caused by lower expression of ICAM- might be responsible, in part, for the muted contact sensitivity in the depigmented animals.17

The Fowl Chicken The pathogenesis of the Fowl chicken model combines a genetic abnormality and an environmental factor.18 Feather melanocytes in mutant chickens die prematurely in vivo and live in vitro; thus an environmental factor in the feather precipitates their death. The superoxide dismutase activity and the glutathione concentration are reduced in the mutant feathers when compared to the wild type. Based on these results, the hypothesis is that premature avian melanocyte death is due to low antioxidant levels of protection and is precipitated by



the accumulation of oxygen radicals in the poorly vascularized feather with a low turnover of tissue fluids.

The B-light


In the B-light mice, the tips of the hairs are pigmented, but the basesare not, because follicular melanocytes die prematurely. I9 This phenotype is the consequence of the dominant light mutation at the brown locus that encodes TRP-1, an enzyme involved in melanin synthesis. This point mutation changes an arginine residue close to the signal sequence to cysteine. As a consequence of the lack of functional TRP-1 in melanosomes, it is likely that a large proportion of the melanin precursors leaks out to the cytoplasm. It is suggested that pigment-cell death results from the inherent toxicity of melanin precursors. Thus, the B-light mice may be an animal model of familial vitiligo due to a dominant mutation throught autotoxicity.

The bcl-2-l-


Three lines of bcl-22- mice have been reported that presented hair hypopigmentation.*0-22 The hair color of these animals appears to be normal during the first hair cycle after birth, and it turns gray during the second hair cycle. This decreased color tone is due to the disappearance of melanocytes and the resulting lack of melanin granules in most of the anagen hair follicles.23 Thus, bcl-2 is essential for the maintenance of ap ropriate life span of melanocytes. In addition, bcl-2- P- mice revealed similar pleiotropic abnormalities, including fulminant apoptosis of lymphoid cells, polycystic kidney, and distorted small intestine. Animal models taken together show that several mechanisms may be involved in vitiligo; further, it is important to emphasize that major differences exist between animal models of vitiligo.

Vitiligo: An Autoimmune Autoantibodies?


Due To

Several autoimmune disorders characterized by autoantibodies to different specific organs have been commonly associated with vitiligo.3 Disorders of the thyroid gland are significantly associated with vitiligo; whereas the value of the associations between vitiligo and pernicious anemia, Addison’s disease, diabetes mellitus, and alopecia areata is still being discussed. Conversly, the only autoantibodies, whose incidence is consistently elevated in vitiligo as compared with controls are those to thyroglobulin and to microsomes. The significance of autoantibodies to nonpigmented cells is probably limited. They probably do not play a direct role in the pathogenesis of vitiligo. Autoantibodies against melanocytes have been convincingly detected in the sera of vitiligo patients by different techniques by several independent groups,24J5

however, antibodies from vitiligo sera are directed in part to antigens specific for pigment cells, but they also are directed to normal tissue antigens, expressed by different cells, in particular keratinocytes.26-28 In addition, antimelanocyte antibodies can be found at low titer in the sera of a small percentage of patients without vitiligo; furthermore, a significant percentage (25-30%) of patients with vitiligo do not exhibit the presence of antimelanocyte antibodies. Recently, it has been shown that tyrosinase, the key enzyme of melanogenesis, is a principal target of antimelanocyte antibodies during vitiligo.29 Serum antibodies against tyrosinase, a 69-75 kD protein, were detected in 77% of vitiligo patients, 0% of normal controls, and 12% of patients with autoimmune endocrine disease without vitiligo or a history of vitiligo. The possibility that levels of antityrosinase antibodies were correlated with vitiligo was proposed as a plausible explanation for the significant percentage of vitiligo patients without antityrosinase antibodies, Several studies concluded that the sera titers of antimelanocyte antibodies were correlated with the activity of vitiligo. They suggested also that a correlation exists between antibody levels and the extent of depigmentation;““.?’ however, a follow-up of patients with active vitiligo showing that the levels of antibodies decrease when the disease is inactive would be a more definite proof. Besides, variations in antimelanocyte antibody levels might be rather secondary to the disease activity and the destruction of melanocytes than their cause. In vitro, vitiligo sera have the ability to destroy melanocytes by complement-mediated cytotoxicity and by antibody-dependent cellular cytotoxicity;32 however, whether the selective killing of melanocytes occurs because vitiligo antibodies are directed to specific melanocyte antigens or because melanocytes are more susceptible to immune injury than other cells remains questionable.“’ Direct immunofluorescence findings are usually negative, with an absence of immunoglobulin and complement deposition, except in one study in which sparce deposits of IgG at the dermal-epidermal junction have been reported in approximately 50% of vitiligo lesions;‘4 however, the sensitivity of immunofluorescence is low, and it might be insufficient to detect the presence of immunoglobulin and complement directed specifically against melanocytes. Vitiliginous human skin grafted onto nude mice becomes repigmented following transplantation,35 which suggests that a primarily melanocyte dysfunction is not involved in the etiology of the depigmenting process. The IgG fraction of serum from vitiligo patients may induce in viva destruction of human melanocytes in normal skin grafted onto nude mice. This study, which mimics the in \,ivo situation, strongly supports, but

does not entirely demonstrate, that ,jutoantibodies found in patients with vitiligo play a pathogenic role or take part in melanocyte damage and are not an epiphenomenon.“h Vitiligo-like leukoderma associated i\,ith melanoma remains an enigmatic phenomenon, observed in particular following interleukin-2 therapy for melanoma patients.37-“9 Antimelanocyte antibodies present during melanoma and vitiligo bind to similar antigens shared by normal and malignant pigment cells. iC This finding suggests that vitiligo and vitiligo-like leukoderma associated with melanoma, although clinically distinct, may have a common physiopathologic mechmism.

Vitiligo: An Autoimmune Cell Immunity?

Disease Mediated by

In addition to possible antibody invoh~ement, vitiligo might be a T-cell mediated disease, bvhereas natural killer cells certainly do not play a significant role during vitiligo .41 The possibility that vitiligo is “a benign cutaneous lymphoma” was raised to explain the svmmetrical appearance of lesions, due to T \-ells shIectively homing within symmetrical sites of the hod!,, but few data support this theory.42 Other observations may fit the hypothesis of involvement of cell-mediated immunity during vitiligo. In some cases, subtle histologic changes are observed within active vitiligo lesion borders (see prior section on the behavior of melanocytes in vitiligo macules). Lt may be speculated that they are due to T-ceil-mediated autoimmunity. In some patients with inflammatory vitiligo, presence of T cells and macrophages seemed to parallel melanocyte disappearance.4~’ Within periiesional skin, epidermis-infiltrating T cells exhibited an increased CD8/CD4 ratio and an increased expression of cutaneous lymphocyte antigen and interleukin-2 receptor. Keratinocytes as well as melanocytes expressed major histocompatibility complex class II antigens, and inflammation was accompanied by increased tenascin content. These observations are suggestil-e of involvement of local immune reactivity in melanc qtc destruction, but they do not permit differentiation between the immune infiltrates being a result as opposed to being the cause of the disease process. The existence of altered ratios of T-ceil subpopulations in blood, body fluids, and cellular infiltrates is still being discussed. Most studies do not provide the relevant data to fit this possibility.J1.G’ The Lanhgerans cell density within vitiligo skin has been variably reported as decreased, normal, or increased.’ Degenerative changes havt> r71sc1 been reported. It seems that a marked depletion of Lanhgerans cells is observed in lesions of activrt vitiligo, whereas a repopulation occurs within lesions of stable and spontaneouslv repigmenting vitiligo. In aLid itinn to these




quantitative changes, a functional impairment of Lanhgerans cells has also been documented;46 however, the situation regarding the number and functions of Lanhgerans cells during vitiligo is far from being clear. The possibility that melanocytes themselves play a role in skin immune reactions has been emphazised. In vivo expression of MHC class II molecules and ICAMby perilesional vitiligo melanocytes has been demonstrated and may be considered supportive of T-cell mediated immunity.“7 To our knowledge, there is no demonstration of systemic or localized increase or decrease in the tissue levels of cytokines/lymphokines and no published study on the possible preferential use of certain T-cell receptor genes during vitiligo.


A Genetic

Clinics in Duvfntolo~~~y



As 30% of the vitiligo patients have a family history of vitiligo, a hereditary factor seems likely;48 however, one must remember that members of a family are usually exposed to the same environmental factors. The mode of heredity of vitiligo suggests that a polygenic trait is involved, with mutations in different genes implicated to induce a predisposition to vitiligo.49 There are very rare families in which the pattern of vitiligo suggests an autosomal dominant form of tranmission. These families are the exception, not the rule. In different large epidemiologic studies, the transmission of vitiligo from parents to offspring ranged from 3% to 7%. Analysis of the distribution pattern of depigmentation in extended kinships, including primary, secondary, and tertiary family members, confirmed that there is clustering of patients in rare kinships. These data strongly suggest a polygenic trait in most families, possibly involving three or four alleles. Demonstration of an intrinsic defect in vitiligo melanocytes would fit the hypothesis of genetic factors in vitiligo. As compared to controls, cultured vitiligo melanocytes show no significant differences in DNA synthesis, tyrosinase activity and responses to ultraviolet B (UVB); however, when long-term cultures are performed, melanocytes show specific cytolytic alterations.50,51They contain dilated and/or circular rough endoplasmic reticulum on examination with the transmission electron microscope. An inherited or intrinsic defect of melanocytes might be a primary event in the pathogenesis of vitiligo; however, in vitro the defect, if any, is nonlethal. Genetic factors that have been considered as possibly involved include MHC genes, important for antigen presentation. HLA-linkage for vitiligo has been reported in different human populations.52 Although it can support the autoimmune hypothesis, HLA-linkage may also indicate that there are subtypes of vitiligo.53 Other candidate genes are numerous but are far from



identified. The autocytotoxic theory, or self-destruction hypothesis, is based mainly on the demonstration that intermediates of melanin synthesis are toxic to melanocytes.’ An expansion of the autocytotoxic hypothesis is that abnormal responses to growth factor or to MSH might also cause melanocyte loss. In vitro, basic fibroblastic growth factor, epidermal growth factor, endothelin-1 and the leukotrienes LTC4 and LTD4 have been shown to be melanocyte growth factors.sJ Thus, genes encoding for proteins implicated in the control of melanocyte proliferation and differentiation should be studied. The proto-oncogene c-kit encodes the transmembrane tyrosine kinase receptor that has a role in the growth regulation of various cell types including melanocytes. The c-kit protein is present on melanocytes in adult human skin. In perilesional skin of some vitiligo patients, there is a reduction in the numbers of melanocytes expressing this receptor;“” however, the meaning of this feature remains unknown, and it is far from demonstrated that an abnormality of the c-kit plays any role in the pathogenesis of vitiligo. Genes determining premature melanocyte death, such as b-cl2 gene, should be also studied.““,s6


Factors in Vitiligo

Schallreuter’s group proposes that vitiligo is a disease of the entire epidermis with a complex biochemical imbalance that leads to direct inhibition of tyrosinase and melanin bleaching by hydroxyl radicals.57 In normal skin, L-tyrosine is the common substrate for melanin biosynthesis by melanocytes and catecolamines biosynthesis by keratinocytes. L-tyrosine production from L-phenylalanine by phenylalanine hydroxylase is ratelimited by the cofactor (6R)-5,6,7,8 tetrahydrobiopterin (6-BH4). 6-BH4 is recycled from 4a-OH-BH4 by an enzyme, the 4a-hydroxy-BH4 deshydratase. In vitiligo patients, it is suggested that activity of 4a-hydroxy-BH4 deshydratase is low, resulting in a build up of 7-BH4 and of hydrogen peroxide in the epidermis. A result of 7-BH4 build up is inhibition of phenylalanine hydroxylase, leading to a defective synthesis of melanin. Furthermore, catalase activity is very low in the epidermis of vitiligo patients, leading to increase in the toxicity of hydrogen peroxide. Therefore, a sudden burst of hydrogen peroxide can be very cytotoxic and may explain active depigmentation. Finally, Schallreuter’s group suggests that defective catecholamine biosynthesis and increased activity of monoamine oxidase A in the epidermis of vitiligo patients may provide a basis for stress-related vitiligo through the formation of hydrogen peroxide; however, the meaning of these biochemical abnormalities is unclear, and it is not demonstrated that they play a significant role in the pathogenesis of vitiligo.

The Neural


The neural hypothesis is based in the first place on the presence of segmental vitiligo. An ultrastructural study of the dermal nerves was performed recently.58 Subtle ultrastructural differences were observed between biopsies taken from marginal and central parts of vitiliginous skin and nonvitiliginous skin. The most consistent feature, seen in all biopsies from vitiliginous skin, was an increased thickness of the basement membrane of Schwann cells. This change was seen in approximately three quarters of dermal nerves in vitiligo biopsies and in about one quarter of dermal nerves in normal control biopsies. About half of the abnormal dermal nerves showed minor axonal damage, whereas indicators of regeneration predominated in the others; in addition, communication between the nervous system and epidermal melanocytes has been recently proved.i4 Abnormalities reflecting possible neuromediated aberrations in p endorphin and met-enkephalin secretion in vitiligo patients and increased immunoreactivity to neuropeptide Y and vasoactive intestinal peptide in vitiligo skin have been reported.6”,fi1 Although little is known about the effects of neuropeptides on human melanocytes, the nervous system may exert a tonic effect on melanocytes in normal or diseased human skin, especially through calcitonin generelated peptide secretion.“” Yet, the role of the nervous system in the pathogenesis of vitiligo is still undefined.



Viral infections have been implicated in the pathogenesis of multiple autoimmune diseases; some of them, like Hashimoto’s thyroiditis, occur with increased frequency in patients with vitiligo. Vitiligo may occur in patients with AIDS; however, no epidemiologic study has suggested a role for viral infections in the pathogenesis of vitiligo. In a recent study,62 polymerase chain reaction was used to detect proviral DNA and DNA genomes of herpes simplex, varicella-zoster, Epsteinbarr, human cytomegalovirus (CMV), HIV, and human T-lymphotropic \,irus in the depigmented skin and uninvolved skin of 29 vitiligo patients, as well as in the skin of 10 healthy persons and 12 persons comprising a spectrum ot other skin diseases. CMV DNA was detected in the depigmented and univolved skin of 38% of vitiligo patients studied and in 0% of control subjects. CMV DNA was especially found in biopsy specimens of progressive vitiligo of relatively brief duration. Search for other viral genomes was negative. These results suggest that some cases of vitiligo might be triggered by viral infection; however, additional studies are needed to confirm these preliminary results and to clarify the role of CMV in the pathogenesis of vitiligo.


of Repigmentation

in Vitiligo

Some vitiligo patients show spontaneoiis repigmentation, but most patients have permanent melanocyte loss. About 50% of these patients will repigment with photochemotherapy. Repigmentation usually occurs in a perifollicular pattern, suggesting that lallicular melanocytes colonize vitiliginous skin. In :nctbt I:X~S of repigmenting vitiligo, studies also arguk; for Clproliferation of follicular melanocytes, followed hy their m&r+ tion;‘,’ however, less commonly, repigmentation might occur from residual intraepidermal mel,mttc~~t~~s. Based on the perifollicular pattern of r,epig&entation, the existence of a melanocyte reservoir in human hair follicles has been postulated. Recently, the tlxlstence of a population of intraepithelial ceils that have immunophenotypic characteristics of rn&urcl mclanocytes within the upper epithelial regions, but iack thll differentiated melanocytic phenotype within the deeper follicular regions, has been demonstrated.‘~ Tht+;c KIT (t ), BCL-2 (+) and TRP-I (-) cells ma! zonqtitutr the precursor melanocyte reservoir of human ?kin. During vitiligo repigmentation, mela:~ocytcs migrate from the outer root sheath of the follicle to the basal layer of epidermis just above the basement membrane. Because keratinocytes are attached together through desmosomes and to the basement m~mhrane by hemidesmosomes, migration of mrlanocytes supposes several complex processes that are not \‘ct understood. Action of mediators that promote the m&ration of pigment cells is required, followed by intr‘rac-tions of melanocytes with extracellular matrix rn~~l~~culrs,.lnd ct>ll surface molecules.“” The effects of melanocyte mitogens ori rnelanocyte migration have been studied recentI>,.’ It \v;i~: demonstrated in Boyden chamber checkerboard an,llysis that bFGF, leukotriene C3 and endothelin-i :vere chemotoctic. They produced directional migratitln. ‘Jhese 3 factors and stem cell factor induced melanocyte chemokinetic movement, with melanoc!;te% rn~n~lng in a random, nonlinear fashion. Transforming growth factor (Ymay also induce a melanocyte chemohinrtic response. Normal melanocytes express mainly (tVp1 and ~$31 integrin receptors and moderate levt,Ls r?f [email protected] and [email protected] integrin receptors. Migration oi i.luman melanocytes is enhanced on a matrix ot collagen IV and is blocked by antibodies to [email protected] and (~3131integrin rpceptors. There is almost no data on prott,.isc secretion by normal melanocytes. hl In contrast, it ib wcil-t>btablished that melanoma cells may secretr pr~tkt‘~1~1:~.

Conclusions The present time is one of the more excjting periods for vitiligo and melanocyte research. While additional informations have been gained, man) new questions have also been raised. Established \.itiligrr is due to the




destruction of melanocytes. A great deal of evidence supports the model in which autoimmunity, in particular that mediated by autoantibodies, is implicated in the vitiliginous depigmentation: autoantibodies are directed specifically against melanocyte antigens, in particular tyrosinase; they have the ability to kill melanocytes in vitro; their level correlates more or less with disease activity; and they are able to induce depigmentation of normal human skin grafted onto nude mice. The possibility that antimelanocyte antibodies are an epiphenomenon secondary to melanocyte destruction and do not play an important pathogenic role is still under investigation. Besides, there is significant evidence that many different biologic factors other than autoimmunity alone contribute to the pathogenesis of vitiligo. Inhibition of melanocyte function and pigment cell destruction occur in a complex epidermal environment. Various factors may combine to produce damage in genetically predisposed melanocytes. These include production of growth factors and cytokines by keratinocytes; antioxidant defenses; oxidative stress; inflammatory mediators; and exogeneous factors, such as ultraviolet radiation, neural stimulation, leukocytes, cytotoxic lymphocytes, and antibodies. Pathways to future therapeutic options have been opened by the identification of these previously unexpected targets involved in melanocyte destruction and/or migration.

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Clinics in Dertnntolo~y



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