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Apoptosis of melanocytes in vitiligo results from antibody penetration
Journal of Autoimmunity 29 (2007) 281e286 www.elsevier.com/locate/jautimm
Review
Apoptosis of melanocytes in vitiligo results from antibody penetration Alejandro Ruiz-Argu¨elles a,*, Gustavo Jime´nez Brito b, Paola Reyes-Izquierdo a, Beatriz Pe´rez-Romano a, Sergio Sa´nchez-Sosa c a
Department of Immunology, Laboratorios Clı´nicos de Puebla, Diaz Ordaz 808, Puebla, PUE 72530, Mexico Colegio Poblano de Dermatologı´a, Universidad Popular Auto´noma del Estado de Puebla, Puebla, Mexico c Department of Pathology, Universidad Popular Auto´noma del Estado de Puebla, Puebla, Mexico
b
Abstract Vitiligo is a rather common disease characterized by depigmentation of skin and mucosae due to the loss of melanocytes, most likely as a result of autoimmune phenomena. In this study we demonstrated apoptotic markers in residual melanocytes in skin biopsies of patients with vitiligo, as well as the presence of serum antibodies to melanocyte-specific antigens in the vast majority of patients. Moreover, we were able to prove that serum IgG antibodies from vitiligo patients, but not from healthy controls, were capable to penetrate into cultured melanocytes in vitro, and trigger them to engage in apoptosis. Our results are consonant with the theory that melanocyte-specific antibodies are responsible for the deletion of melanocytes through antibody penetration and apoptosis. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Cell penetrating antibodies; Vitiligo
1. Introduction Vitiligo is a rather common pigmentation disorder in which melanocytes in the skin, mucous membranes and the retina are destroyed. As a result, white patches of skin appear on different parts of the body. The hair that grows in areas affected by vitiligo usually turns white. Several theories have been forwarded to explain the cause of vitiligo, and a most plausible one is the autoimmune origin of the disease [1e4]. Antibodies to different antigens in melanocytes are present in the serum of a large proportion of patients as compared to healthy individuals and auto reactive T cells have been demonstrated in some affected subjects. Additionally, vitiligo seems to be more common in people with certain autoimmune diseases such as hyperthyroidism, adrenocortical insufficiency, alopecia areata and pernicious anemia, and finally, the use of topical immunosuppressant therapy has proved useful not only to detain the progression of the disease, but to recover pigmentation to
a certain degree [5e14]. Inasmuch as depigmentation of the skin is not encompassed by an inflammatory response, it has been proposed that melanocyte death in vitiligo is due to apoptosis [15], a phenomenon that has been previously shown to be triggered by the penetration of certain antibodies in other cells and cell lines [16e18]. About 40 to 50 million people worldwide have vitiligo and, although in its primary form it is not life threatening, the cosmetic and psychological effects of the disease demand for an effective therapy, which has to rely on a better understanding of its pathogenesis. Herein, we show data that strongly support that self-reactive antibodies are capable of penetrating and deleting melanocytes by triggering apoptosis of these cells. These data should be compared with other critical issues on loss of tolerance in other autoimmune diseases [19e27]. 2. Material and methods 2.1. Patients
* Corresponding author. Tel.: þ52 222 243 8100; fax: þ52 222 243 8428. E-mail address: [email protected] (A. Ruiz-Argu¨elles). 0896-8411/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2007.07.012
Fifteen patients with vitiligo were accrued in the study. The elapsed time since diagnosis ranged from 1 to 24 months with
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a median of 19 months. Twelve patients were female and their ages ranged from 9 months to 53 years, with a median of 29 years. Five of them had never received treatment for this disease, while the remaining 10 had received no specific treatment during the last 12 months. Ten normal individuals (8 female) with the same age median were studied as a control group. 2.2. Samples After previous written consent, blood was drawn from each subject into EDTAK3 containing tubes for cell analysis and into serum separating tubes (SSTÒ, Becton Dickinson) for serological studies. Additionally a punch skin biopsy from the border of the lesions was performed in all patients. Biopsies and serum samples were stored frozen at 170 C and 20 C, respectively, until tested. Blood cell studies were done immediately. 2.3. Cell analysis Proportions and absolute numbers of total B, T and NK cells, as well as cytotoxic T (Tc), helper T (Th) and regulatory T (Treg) cells in peripheral blood were determined by multiparameter flow cytometry. Whole blood samples were stained with two admixtures of fluorescent antibodies; one tube contained antibodies to CD45 (FITC), CD56 (RD1), CD19 (ECD), CD3 (PC5), and CD8 (PC7) and the other had antibodies to CD25 (FITC), CD4 (PE) and CD3 (APC); all antibodies were purchased from Coulter-Immunotech. After hemolysis and fixation, cells were analyzed in a Cytomics FC500Ò (Beckman Coulter) instrument equipped with an Argon (blue) and a Helium-Neon (red) laser [17]. Absolute numbers of cells were derived from the total white blood cell counts and differential performed in a hematology analyzer (HmXÒ, Beckman Coulter). 2.4. Detection of melanocyte-specific antibodies This was performed by Western blot assay. Small samples of skin obtained from the nipple of a surgical specimen were minced, soluble extracts prepared in phosphateesodiumdodecyl-sulfate and 2-mercaptoethanol buffer as described [31], and electrophoresed on 11% SDSepolyacrylamide gels [32]. The proteins were then electrotransferred to nitrocellulose sheets, and strips from these were exposed to diluted serum samples from all subjects. The binding of antibodies was evidenced using a commercial biotinestreptavidineperoxidase system (Vectastain ABCÒ kit, Elite). Approximate molecular weights of reactive fractions were estimated by commercial standards that were simultaneously electrophoresed and stained with amido black after electrotransferred to nitrocellulose. 2.5. Demonstration of apoptotic markers in melanocytes This was performed in cryosections of the patients skin biopsies by immunohistochemical staining. Antibodies to apoptosis peptidase activating factor (Apaf-1) (Neo Markers), Bcl-2
(Dako) and Caspase 9 (Neo Markers) were used to detect these proteins in tissue sections. To ascertain that the abovementioned markers were in fact expressed by melanocytes, all preparations were simultaneously stained with the HMB45 monoclonal antibody to gp-100 (Dako), a glycoprotein whose expression is restricted to melanocytes and melanoma cells. Samples were processed with the aid of an automated system (DakoautostainerÒ, Dako) and developed with a commercial alkaline-phosphatase-polymer system (UltraVision Labeled PolymerÒ, Labvision) and fast-red as chromogen (Fast RedÒ, Labvision). Finally, specimens were counterstained with hematoxylin and mounted in glycine. Tissue sections from a malignant melanoma, seminiferous tubules, follicular lymphoma and normal skin were used as positive controls for the HMB-45, Apaf1, Bcl-2 and Caspase 9 antibodies, respectively [33,36e39]. 2.6. In vitro induction of apoptosis of melanocytes by antibodies The serum gamma-globulin fraction from six patients and six controls was enriched by repeated (3) ammonium sulfate precipitation and further purified by diethyl-amino-ethylcellulose ion-exchange chromatography. After extensive dialysis, these fractions were used to induce apoptosis of cultured melanocytes as described below. Primary cultures of melanocytes were established in 24-well flat-bottomed plates (Nunc) from normal skin according to the method described by Redondo [33], using melanocyte growth medium MGM-4Ò (Cambrex). Confluence was reached at 14 to 16 days after incubation at 37 C in a 5% CO2 and 100% relative humidity atmosphere. From the very early stages of the cultures, melanocytes presented their characteristic dendritic morphology that remained unchanged until they were harvested (at confluence) for the experiments. The purified immunoglobulin fractions from patients’ and controls’ serum were added to duplicate wells during the last 24 h of culture at a final protein concentration of 2.5 mg/ml and harvested by scraping with a rubber policeman. Cells were washed with phosphate-buffered saline, pelleted by centrifugation in conical tubes and their total nuclear DNA stained with a commercial reagent (DNA-PrepÒ, Beckman Coulter) consisting of a non-ionic detergent and an admixture of propidium iodide and ribonuclease. After incubation, cells were analyzed by flow cytometer in a Cytomics FC-500 instrument, using the peak and integral fluorescence signals at 675 nm to gate out cell doublets [17]. The raw histograms from all samples were further analyzed with the aid of specialized software (MulticycleÒ Phoenix Flow Systems) to enumerate the proportions of cells which had lost low molecular weight DNA (sub G0/G1 peak) as indicative of late apoptosis. 2.7. Demonstration of in vitro antibody penetration into melanocytes Aliquots of cultured melanocytes exposed to the gammaglobulin fractions of controls’ and patients’ serum were
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extensively washed, resuspended in 100 ml of 0.1% saponin in Hanks’ balanced salt solution (HBSS) and incubated with a fluorescein-conjugated anti-human immunoglobulin antiserum (Antibodies Inc., Davis, CA) for 20 min. After completion of this incubation, cells were washed once more with PBS without saponin, resuspended in 1 ml of PBS and analyzed by flow cytometry. A few seconds prior to analysis, 10 ml/ml of a saturated solution of crystal violet was added to quench surface fluorescence and detect only intracellular emissions. Results were expressed as percent fluorescent cells, using as a negative control cells that were simultaneously treated in the same fashion but in the absence of saponin. 2.8. Statistical analysis
283
205,000
116,000
97,400
66,000
Comparison of mean values of cell populations and linear regression analysis were performed with the aid of MedCalcÒ statistical software.
45,000 29,000
3. Results 3.1. Lymphoid cell subpopulations Since T regulatory (Treg) cells appear to play an important role in suppressing normal immune responses, we have here evaluated the potential role of Treg cells in immune dysfunction in vitiligo; however, no differences were found between patients and controls in the relative or absolute numbers of total lymphocytes, CD19þ (B), CD3þ (T), CD3þ/CD4þ (Th), CD3þ/CD8þ (Tc), CD3þ/CD4þ/CD25þ (Treg) and CD3/ CD56þ (NK) cells. Table 1 summarizes these findings. 3.2. Serum antibodies to melanocytes Fig. 1 is the result of one experiment showing the reactivity of serum antibodies in Western blots. Table 2 summarizes results from all experiments. Serum samples from 14/15 patients showed antibodies to at least two of the protein bands in the Western blot assays, while only 1/10 controls showed reaction with a single protein. Bands corresponding to the molecular weight of immunoglobulin heavy (z50 kDa) and
Table 1 Peripheral blood lymphocyte subpopulations in patients with vitiligo and normal controls Cell subpopulations
B (CD19þ) Total T (CD3þ) Helper T (CD3þ/CD4þ) Regulatory T (CD3þ/CD4þ /CD25þ) Cytotoxic T (CD3þ/CD8þ) NK (CD3/CD56þ)
Patients
Controls
%
No. per ml
%
No per ml
83 82 9 56 7 14 4
158 23 1745 198 1096 167 295 74
93 79 10 54 9 17 6
170 27 1678 181 991 178 318 99
25 6 10 4
540 87 214 23
24 4 12 5
512 61 234 34
Values are expressed as mean standard deviation.
Fig. 1. Western blots of sera from patients with vitiligo showing the reactivity of IgG antibodies with different electrophoretic fractions of an extract of a melanocyte-rich skin specimen.
light (z23 kDa) chains were persistently observed, even in the absence of serum, due to the contamination of the skin specimen with blood. The most consistent reaction was seen against a 75 kDa protein, which most likely corresponds to tyrosinase, the most commonly found antigenic target of vitiligo autoantibodies. Candidate antigens for the other reactions are transferrin receptor, C-kit, Lamp-2, Vit-90, Vit-75 and Vit-40.
3.3. Apoptotic markers in melanocytes of vitiligo skin biopsies All biopsies from patients showed spongiosis and reduction of the number of melanocytes to a certain degree, according to the area of skin under observation. As expected, melanocytes were practically absent in fully depigmented zones, while they were reduced in the borders of these lesions. Adjacent normal skin, however, contained diminished numbers of melanocytes than normal skin. All the specimens showed lymphocyte infiltrates that, most strikingly, were located in juxtaposition to areas of overt depigmentation while not in those where residual melanocytes were present. Staining for Bcl-2 revealed that infiltrates consisted mainly of B lymphocytes. Melanophages were commonly seen in these zones, while sweat glands and hair follicles were normally conserved. Approximately 10e20% of remaining melanocytes in the border of the lesions of 11/15 biopsies showed expression of Bcl-2, while very few, if any, showed Apaf-1 or caspase 9,
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284
Table 2 Positive reactions of vitiligo patients’ and controls’ sera with the different electrophoretic fractions of an extract of human melanocytes Electrophoretic fractions, kDa 210 Patients 1 þ 2 þ 3 4 5 þ 6 7 þ 8 þ 9 10 11 12 13 þ 14 15 Controls 1 2 3 4 5 6 7 8 9 10
176
151
123
92
90
89
85
75
42
þ þ þ
þ
þ þ þ þ
þ
þ
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ þ þ
þ þ
þ þ þ
þ
þ þ
þ þ þ
þ
þ
þ
þ þ
Fig. 3. Bcl-2 expression in residual melanocytes in an area adjacent to a depigmented lesion.
3.4. In vitro induction of apoptosis by IgG from patients with vitiligo
þ
(þ) Indicates a positive reaction with the corresponding fraction.
No morphological changes were evident in melanocyte cultures exposed neither to IgG from vitiligo patients, nor to IgG from normal individuals; however, DNA content analysis showed a significant difference in the proportions of cells that incurred in apoptosis when exposed to vitiligo IgG (32.16 6.24%) as compared to those that were exposed to normal IgG (1.33 0.82%; t ¼ 12.92, p < 0.0001). 3.5. Antibody penetration in melanocytes
which were both abundantly expressed on keratinocytes. This apparent controversy will be discussed later. Figs. 2 and 3 are examples of skin biopsies stained with antibodies to Bcl-2 and HMB-45.
Intracellular IgG was found in a much larger proportion of cells exposed to vitiligo IgG (62.66 8.68%) than in cells exposed to normal IgG (1.83 1.17%; t ¼ 17.074, p < 0.0001). Fig. 4 shows the individual values and correlation between the cells that showed antibody penetration and apoptosis after exposure to IgG obtained from the serum of 6 patients with vitiligo. 4. Discussion
Fig. 2. Lymphocyte infiltrate (solid oval) in juxtaposition to a depigmented area of the skin (dotted oval). The arrow indicates the reactivity of MoAb HMB-45 in an adjacent area where abundant melanocytes are still present.
Taken together, our results are consonant with the hypothesis that antibodies directed to melanocyte-specific antigens may penetrate into these cells and trigger them to undergo into apoptosis. In this study, we confirmed the presence of serum antibodies to melanocytes in the vast majority of the patients, while they were practically absent in the control group. Despite the lack of conclusive evidence, there are several facts that favor the fact that melanocyte deletion in vitiligo results from apoptosis, particularly the finding of melanophagia and the absence of an inflammatory response. Our demonstration that residual melanocytes in biopsies from vitiligo patients express apoptotic markers, strongly support this notion [15]. Although apparently controversial, the predominance of anti-apoptotic markers
A. Ruiz-Argu¨elles et al. / Journal of Autoimmunity 29 (2007) 281e286 42 40
Apoptosis, %
38 36 34 32 30 28 26 24 45
50
55
60
65
70
75
285
total serum IgG and not purified antibodies. Instead, we used a large excess of IgG and exposed the cultured cells for the time and temperature known from other studies to be optimal for antibody penetration [18,27e30,34]. Although vitiligo is not by itself a life-threatening disease, it has sometimes devastating cosmetic, psychological and social consequences. Many of the therapeutic approaches that have been tried in this disease have no scientific rationale and, according to our results, the use of corticosteroid-containing ointments might even be harmful. We believe that our findings might contribute to the design of new therapeutic strategies for this disease, and appear to explain the favorable response that these patients show to the use of topical immunosuppressants [47,48]. The data presented herein can have an interesting contrast and comparison with related papers in this issue [49e62].
Penetration, % Fig. 4. Linear regression between antibody penetration and apoptosis of melanocytes exposed to the enriched IgG fractions from the serum of 6 patients with vitiligo.
(Bcl-2) over pro-apoptotic ones (Apaf-1 and caspase 9) in these residual melanocytes may reflect that the activation of antiapoptotic genes in certain cells is, indeed, responsible for their survival. Perhaps those cells where the expression of proapoptotic genes was predominant incurred rapidly in apoptosis and thence are no longer found as residual melanocytes. A similar finding and explanation has been described for lymphocytes undergoing apoptosis after penetration of anti-dsDNA monoclonal antibodies [29]. The expression of Apaf-1 and caspase 9 in a small fraction of residual melanocytes supports the fact that apoptosis is induced by antibody penetration, for it has been described in other publications that penetration of antibodies triggers mainly the negligent pathway of apoptosis, and only seldom involves membrane signals [29,46]. The presence of B lymphocyte infiltrates in juxtaposition to depigmented zones of the affected skin is also consonant with the idea that the autoimmune phenomenon in this disease is mediated predominantly by humoral mediators; but it also buttresses the possibility that such reactions are localized or enclosed to certain compartments of the skin. This feature suggests that melanocyte depletion is mediated by locally secreted, rather than circulating antibodies, and explains the patchy distribution of the lesions, instead of a generalized depigmentation of skin and mucosae. This ‘‘enclosure’’ might also explain the normalcy of lymphocyte subpopulations in peripheral blood, particularly of Treg cells, that we found in this study. It is possible to further sustain the pathogenic role of antimelanocyte antibodies in the selective deletion of melanocytes in vitiligo, by the demonstration that IgG from patients with vitiligo, but not from normal individuals under the same experimental conditions, were capable in vitro to penetrate into cultured melanocytes and trigger apoptosis. The correlation of the proportion of cells where penetration of IgG was observed with that of cells that incurred in apoptosis, is suggestive that the latter might be induced by the former. We did not attempt to perform dose response experiments since we used
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Review
Apoptosis of melanocytes in vitiligo results from antibody penetration Alejandro Ruiz-Argu¨elles a,*, Gustavo Jime´nez Brito b, Paola Reyes-Izquierdo a, Beatriz Pe´rez-Romano a, Sergio Sa´nchez-Sosa c a
Department of Immunology, Laboratorios Clı´nicos de Puebla, Diaz Ordaz 808, Puebla, PUE 72530, Mexico Colegio Poblano de Dermatologı´a, Universidad Popular Auto´noma del Estado de Puebla, Puebla, Mexico c Department of Pathology, Universidad Popular Auto´noma del Estado de Puebla, Puebla, Mexico
b
Abstract Vitiligo is a rather common disease characterized by depigmentation of skin and mucosae due to the loss of melanocytes, most likely as a result of autoimmune phenomena. In this study we demonstrated apoptotic markers in residual melanocytes in skin biopsies of patients with vitiligo, as well as the presence of serum antibodies to melanocyte-specific antigens in the vast majority of patients. Moreover, we were able to prove that serum IgG antibodies from vitiligo patients, but not from healthy controls, were capable to penetrate into cultured melanocytes in vitro, and trigger them to engage in apoptosis. Our results are consonant with the theory that melanocyte-specific antibodies are responsible for the deletion of melanocytes through antibody penetration and apoptosis. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Cell penetrating antibodies; Vitiligo
1. Introduction Vitiligo is a rather common pigmentation disorder in which melanocytes in the skin, mucous membranes and the retina are destroyed. As a result, white patches of skin appear on different parts of the body. The hair that grows in areas affected by vitiligo usually turns white. Several theories have been forwarded to explain the cause of vitiligo, and a most plausible one is the autoimmune origin of the disease [1e4]. Antibodies to different antigens in melanocytes are present in the serum of a large proportion of patients as compared to healthy individuals and auto reactive T cells have been demonstrated in some affected subjects. Additionally, vitiligo seems to be more common in people with certain autoimmune diseases such as hyperthyroidism, adrenocortical insufficiency, alopecia areata and pernicious anemia, and finally, the use of topical immunosuppressant therapy has proved useful not only to detain the progression of the disease, but to recover pigmentation to
a certain degree [5e14]. Inasmuch as depigmentation of the skin is not encompassed by an inflammatory response, it has been proposed that melanocyte death in vitiligo is due to apoptosis [15], a phenomenon that has been previously shown to be triggered by the penetration of certain antibodies in other cells and cell lines [16e18]. About 40 to 50 million people worldwide have vitiligo and, although in its primary form it is not life threatening, the cosmetic and psychological effects of the disease demand for an effective therapy, which has to rely on a better understanding of its pathogenesis. Herein, we show data that strongly support that self-reactive antibodies are capable of penetrating and deleting melanocytes by triggering apoptosis of these cells. These data should be compared with other critical issues on loss of tolerance in other autoimmune diseases [19e27]. 2. Material and methods 2.1. Patients
* Corresponding author. Tel.: þ52 222 243 8100; fax: þ52 222 243 8428. E-mail address: [email protected] (A. Ruiz-Argu¨elles). 0896-8411/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2007.07.012
Fifteen patients with vitiligo were accrued in the study. The elapsed time since diagnosis ranged from 1 to 24 months with
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a median of 19 months. Twelve patients were female and their ages ranged from 9 months to 53 years, with a median of 29 years. Five of them had never received treatment for this disease, while the remaining 10 had received no specific treatment during the last 12 months. Ten normal individuals (8 female) with the same age median were studied as a control group. 2.2. Samples After previous written consent, blood was drawn from each subject into EDTAK3 containing tubes for cell analysis and into serum separating tubes (SSTÒ, Becton Dickinson) for serological studies. Additionally a punch skin biopsy from the border of the lesions was performed in all patients. Biopsies and serum samples were stored frozen at 170 C and 20 C, respectively, until tested. Blood cell studies were done immediately. 2.3. Cell analysis Proportions and absolute numbers of total B, T and NK cells, as well as cytotoxic T (Tc), helper T (Th) and regulatory T (Treg) cells in peripheral blood were determined by multiparameter flow cytometry. Whole blood samples were stained with two admixtures of fluorescent antibodies; one tube contained antibodies to CD45 (FITC), CD56 (RD1), CD19 (ECD), CD3 (PC5), and CD8 (PC7) and the other had antibodies to CD25 (FITC), CD4 (PE) and CD3 (APC); all antibodies were purchased from Coulter-Immunotech. After hemolysis and fixation, cells were analyzed in a Cytomics FC500Ò (Beckman Coulter) instrument equipped with an Argon (blue) and a Helium-Neon (red) laser [17]. Absolute numbers of cells were derived from the total white blood cell counts and differential performed in a hematology analyzer (HmXÒ, Beckman Coulter). 2.4. Detection of melanocyte-specific antibodies This was performed by Western blot assay. Small samples of skin obtained from the nipple of a surgical specimen were minced, soluble extracts prepared in phosphateesodiumdodecyl-sulfate and 2-mercaptoethanol buffer as described [31], and electrophoresed on 11% SDSepolyacrylamide gels [32]. The proteins were then electrotransferred to nitrocellulose sheets, and strips from these were exposed to diluted serum samples from all subjects. The binding of antibodies was evidenced using a commercial biotinestreptavidineperoxidase system (Vectastain ABCÒ kit, Elite). Approximate molecular weights of reactive fractions were estimated by commercial standards that were simultaneously electrophoresed and stained with amido black after electrotransferred to nitrocellulose. 2.5. Demonstration of apoptotic markers in melanocytes This was performed in cryosections of the patients skin biopsies by immunohistochemical staining. Antibodies to apoptosis peptidase activating factor (Apaf-1) (Neo Markers), Bcl-2
(Dako) and Caspase 9 (Neo Markers) were used to detect these proteins in tissue sections. To ascertain that the abovementioned markers were in fact expressed by melanocytes, all preparations were simultaneously stained with the HMB45 monoclonal antibody to gp-100 (Dako), a glycoprotein whose expression is restricted to melanocytes and melanoma cells. Samples were processed with the aid of an automated system (DakoautostainerÒ, Dako) and developed with a commercial alkaline-phosphatase-polymer system (UltraVision Labeled PolymerÒ, Labvision) and fast-red as chromogen (Fast RedÒ, Labvision). Finally, specimens were counterstained with hematoxylin and mounted in glycine. Tissue sections from a malignant melanoma, seminiferous tubules, follicular lymphoma and normal skin were used as positive controls for the HMB-45, Apaf1, Bcl-2 and Caspase 9 antibodies, respectively [33,36e39]. 2.6. In vitro induction of apoptosis of melanocytes by antibodies The serum gamma-globulin fraction from six patients and six controls was enriched by repeated (3) ammonium sulfate precipitation and further purified by diethyl-amino-ethylcellulose ion-exchange chromatography. After extensive dialysis, these fractions were used to induce apoptosis of cultured melanocytes as described below. Primary cultures of melanocytes were established in 24-well flat-bottomed plates (Nunc) from normal skin according to the method described by Redondo [33], using melanocyte growth medium MGM-4Ò (Cambrex). Confluence was reached at 14 to 16 days after incubation at 37 C in a 5% CO2 and 100% relative humidity atmosphere. From the very early stages of the cultures, melanocytes presented their characteristic dendritic morphology that remained unchanged until they were harvested (at confluence) for the experiments. The purified immunoglobulin fractions from patients’ and controls’ serum were added to duplicate wells during the last 24 h of culture at a final protein concentration of 2.5 mg/ml and harvested by scraping with a rubber policeman. Cells were washed with phosphate-buffered saline, pelleted by centrifugation in conical tubes and their total nuclear DNA stained with a commercial reagent (DNA-PrepÒ, Beckman Coulter) consisting of a non-ionic detergent and an admixture of propidium iodide and ribonuclease. After incubation, cells were analyzed by flow cytometer in a Cytomics FC-500 instrument, using the peak and integral fluorescence signals at 675 nm to gate out cell doublets [17]. The raw histograms from all samples were further analyzed with the aid of specialized software (MulticycleÒ Phoenix Flow Systems) to enumerate the proportions of cells which had lost low molecular weight DNA (sub G0/G1 peak) as indicative of late apoptosis. 2.7. Demonstration of in vitro antibody penetration into melanocytes Aliquots of cultured melanocytes exposed to the gammaglobulin fractions of controls’ and patients’ serum were
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extensively washed, resuspended in 100 ml of 0.1% saponin in Hanks’ balanced salt solution (HBSS) and incubated with a fluorescein-conjugated anti-human immunoglobulin antiserum (Antibodies Inc., Davis, CA) for 20 min. After completion of this incubation, cells were washed once more with PBS without saponin, resuspended in 1 ml of PBS and analyzed by flow cytometry. A few seconds prior to analysis, 10 ml/ml of a saturated solution of crystal violet was added to quench surface fluorescence and detect only intracellular emissions. Results were expressed as percent fluorescent cells, using as a negative control cells that were simultaneously treated in the same fashion but in the absence of saponin. 2.8. Statistical analysis
283
205,000
116,000
97,400
66,000
Comparison of mean values of cell populations and linear regression analysis were performed with the aid of MedCalcÒ statistical software.
45,000 29,000
3. Results 3.1. Lymphoid cell subpopulations Since T regulatory (Treg) cells appear to play an important role in suppressing normal immune responses, we have here evaluated the potential role of Treg cells in immune dysfunction in vitiligo; however, no differences were found between patients and controls in the relative or absolute numbers of total lymphocytes, CD19þ (B), CD3þ (T), CD3þ/CD4þ (Th), CD3þ/CD8þ (Tc), CD3þ/CD4þ/CD25þ (Treg) and CD3/ CD56þ (NK) cells. Table 1 summarizes these findings. 3.2. Serum antibodies to melanocytes Fig. 1 is the result of one experiment showing the reactivity of serum antibodies in Western blots. Table 2 summarizes results from all experiments. Serum samples from 14/15 patients showed antibodies to at least two of the protein bands in the Western blot assays, while only 1/10 controls showed reaction with a single protein. Bands corresponding to the molecular weight of immunoglobulin heavy (z50 kDa) and
Table 1 Peripheral blood lymphocyte subpopulations in patients with vitiligo and normal controls Cell subpopulations
B (CD19þ) Total T (CD3þ) Helper T (CD3þ/CD4þ) Regulatory T (CD3þ/CD4þ /CD25þ) Cytotoxic T (CD3þ/CD8þ) NK (CD3/CD56þ)
Patients
Controls
%
No. per ml
%
No per ml
83 82 9 56 7 14 4
158 23 1745 198 1096 167 295 74
93 79 10 54 9 17 6
170 27 1678 181 991 178 318 99
25 6 10 4
540 87 214 23
24 4 12 5
512 61 234 34
Values are expressed as mean standard deviation.
Fig. 1. Western blots of sera from patients with vitiligo showing the reactivity of IgG antibodies with different electrophoretic fractions of an extract of a melanocyte-rich skin specimen.
light (z23 kDa) chains were persistently observed, even in the absence of serum, due to the contamination of the skin specimen with blood. The most consistent reaction was seen against a 75 kDa protein, which most likely corresponds to tyrosinase, the most commonly found antigenic target of vitiligo autoantibodies. Candidate antigens for the other reactions are transferrin receptor, C-kit, Lamp-2, Vit-90, Vit-75 and Vit-40.
3.3. Apoptotic markers in melanocytes of vitiligo skin biopsies All biopsies from patients showed spongiosis and reduction of the number of melanocytes to a certain degree, according to the area of skin under observation. As expected, melanocytes were practically absent in fully depigmented zones, while they were reduced in the borders of these lesions. Adjacent normal skin, however, contained diminished numbers of melanocytes than normal skin. All the specimens showed lymphocyte infiltrates that, most strikingly, were located in juxtaposition to areas of overt depigmentation while not in those where residual melanocytes were present. Staining for Bcl-2 revealed that infiltrates consisted mainly of B lymphocytes. Melanophages were commonly seen in these zones, while sweat glands and hair follicles were normally conserved. Approximately 10e20% of remaining melanocytes in the border of the lesions of 11/15 biopsies showed expression of Bcl-2, while very few, if any, showed Apaf-1 or caspase 9,
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284
Table 2 Positive reactions of vitiligo patients’ and controls’ sera with the different electrophoretic fractions of an extract of human melanocytes Electrophoretic fractions, kDa 210 Patients 1 þ 2 þ 3 4 5 þ 6 7 þ 8 þ 9 10 11 12 13 þ 14 15 Controls 1 2 3 4 5 6 7 8 9 10
176
151
123
92
90
89
85
75
42
þ þ þ
þ
þ þ þ þ
þ
þ
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ þ þ
þ þ
þ þ þ
þ
þ þ
þ þ þ
þ
þ
þ
þ þ
Fig. 3. Bcl-2 expression in residual melanocytes in an area adjacent to a depigmented lesion.
3.4. In vitro induction of apoptosis by IgG from patients with vitiligo
þ
(þ) Indicates a positive reaction with the corresponding fraction.
No morphological changes were evident in melanocyte cultures exposed neither to IgG from vitiligo patients, nor to IgG from normal individuals; however, DNA content analysis showed a significant difference in the proportions of cells that incurred in apoptosis when exposed to vitiligo IgG (32.16 6.24%) as compared to those that were exposed to normal IgG (1.33 0.82%; t ¼ 12.92, p < 0.0001). 3.5. Antibody penetration in melanocytes
which were both abundantly expressed on keratinocytes. This apparent controversy will be discussed later. Figs. 2 and 3 are examples of skin biopsies stained with antibodies to Bcl-2 and HMB-45.
Intracellular IgG was found in a much larger proportion of cells exposed to vitiligo IgG (62.66 8.68%) than in cells exposed to normal IgG (1.83 1.17%; t ¼ 17.074, p < 0.0001). Fig. 4 shows the individual values and correlation between the cells that showed antibody penetration and apoptosis after exposure to IgG obtained from the serum of 6 patients with vitiligo. 4. Discussion
Fig. 2. Lymphocyte infiltrate (solid oval) in juxtaposition to a depigmented area of the skin (dotted oval). The arrow indicates the reactivity of MoAb HMB-45 in an adjacent area where abundant melanocytes are still present.
Taken together, our results are consonant with the hypothesis that antibodies directed to melanocyte-specific antigens may penetrate into these cells and trigger them to undergo into apoptosis. In this study, we confirmed the presence of serum antibodies to melanocytes in the vast majority of the patients, while they were practically absent in the control group. Despite the lack of conclusive evidence, there are several facts that favor the fact that melanocyte deletion in vitiligo results from apoptosis, particularly the finding of melanophagia and the absence of an inflammatory response. Our demonstration that residual melanocytes in biopsies from vitiligo patients express apoptotic markers, strongly support this notion [15]. Although apparently controversial, the predominance of anti-apoptotic markers
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Apoptosis, %
38 36 34 32 30 28 26 24 45
50
55
60
65
70
75
285
total serum IgG and not purified antibodies. Instead, we used a large excess of IgG and exposed the cultured cells for the time and temperature known from other studies to be optimal for antibody penetration [18,27e30,34]. Although vitiligo is not by itself a life-threatening disease, it has sometimes devastating cosmetic, psychological and social consequences. Many of the therapeutic approaches that have been tried in this disease have no scientific rationale and, according to our results, the use of corticosteroid-containing ointments might even be harmful. We believe that our findings might contribute to the design of new therapeutic strategies for this disease, and appear to explain the favorable response that these patients show to the use of topical immunosuppressants [47,48]. The data presented herein can have an interesting contrast and comparison with related papers in this issue [49e62].
Penetration, % Fig. 4. Linear regression between antibody penetration and apoptosis of melanocytes exposed to the enriched IgG fractions from the serum of 6 patients with vitiligo.
(Bcl-2) over pro-apoptotic ones (Apaf-1 and caspase 9) in these residual melanocytes may reflect that the activation of antiapoptotic genes in certain cells is, indeed, responsible for their survival. Perhaps those cells where the expression of proapoptotic genes was predominant incurred rapidly in apoptosis and thence are no longer found as residual melanocytes. A similar finding and explanation has been described for lymphocytes undergoing apoptosis after penetration of anti-dsDNA monoclonal antibodies [29]. The expression of Apaf-1 and caspase 9 in a small fraction of residual melanocytes supports the fact that apoptosis is induced by antibody penetration, for it has been described in other publications that penetration of antibodies triggers mainly the negligent pathway of apoptosis, and only seldom involves membrane signals [29,46]. The presence of B lymphocyte infiltrates in juxtaposition to depigmented zones of the affected skin is also consonant with the idea that the autoimmune phenomenon in this disease is mediated predominantly by humoral mediators; but it also buttresses the possibility that such reactions are localized or enclosed to certain compartments of the skin. This feature suggests that melanocyte depletion is mediated by locally secreted, rather than circulating antibodies, and explains the patchy distribution of the lesions, instead of a generalized depigmentation of skin and mucosae. This ‘‘enclosure’’ might also explain the normalcy of lymphocyte subpopulations in peripheral blood, particularly of Treg cells, that we found in this study. It is possible to further sustain the pathogenic role of antimelanocyte antibodies in the selective deletion of melanocytes in vitiligo, by the demonstration that IgG from patients with vitiligo, but not from normal individuals under the same experimental conditions, were capable in vitro to penetrate into cultured melanocytes and trigger apoptosis. The correlation of the proportion of cells where penetration of IgG was observed with that of cells that incurred in apoptosis, is suggestive that the latter might be induced by the former. We did not attempt to perform dose response experiments since we used
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