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IgG Anti-Melanocyte Antibodies Purified from Patients with Active Vitiligo Induce HLA-DR and Intercellular Adhesion Molecule-1 Expression and an Increase in Interleukin-8 Release by Melanocytes

2000; Elsevier BV; Volume: 115; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2000.00130.x

1523-1747

Yuling Li, Chia‐Li Yu, Hsin‐Su Yu,

Glycosylation and Glycoproteins Research

An immunologic hypothesis is currently proposed as a possible pathogenesis of nonsegmental-type vitiligo. IgG antibodies against melanocyte surface antigens exist in the serum of patients with vitiligo vulgaris. IgG anti-melanocyte antibodies were reported to induce melanocyte damage in vitro by a complement-mediated mechanism and antibody-dependent cellular cytotoxicity. Perilesional melanocytes express major histocompatibility complex class II antigens and a higher intercellular adhesion molecule-1 compared with those in normal skin. The purpose of this study was to determine the role of IgG anti-melanocyte antibodies in the inappropriate expression of major histocompatibility complex class II antigens and intercellular adhesion molecule-1 on melanocytes. IgG anti-melanocyte antibody samples were purified from the individual serum of patients with active vitiligo. After incubation of IgG anti-melanocyte antibodies with cultured melanocytes, the results revealed: (i) IgG anti-melanocyte antibody stimulated HLA-DR expression on melanocytes; (ii) intercellular adhesion molecule-1 expression on melanocytes was significantly induced by IgG anti-melanocyte antibodies; and (iii) IgG anti-melanocyte antibodies induced an increase in interleukin-8 release from melanocytes. The major histocompatibility complex class II molecules expressed in melanocytes can present antigens to CD4 helper cells as antigen-presenting cells and elicit an immune response. Intercellular adhesion molecule-1 is an important adhesion molecule involved in leukocyte and parenchymal cell interaction and thus plays an essential part in immunologic and inflammatory reactions. It is reasonable to speculate that abnormal expressions of HLA-DR and intercellular adhesion molecule-1 on melanocytes by IgG anti-melanocyte antibodies would present vitiligo antigens and allow the antigen-specific immune effector cell attack that results in melanocytotoxicity. An immunologic hypothesis is currently proposed as a possible pathogenesis of nonsegmental-type vitiligo. IgG antibodies against melanocyte surface antigens exist in the serum of patients with vitiligo vulgaris. IgG anti-melanocyte antibodies were reported to induce melanocyte damage in vitro by a complement-mediated mechanism and antibody-dependent cellular cytotoxicity. Perilesional melanocytes express major histocompatibility complex class II antigens and a higher intercellular adhesion molecule-1 compared with those in normal skin. The purpose of this study was to determine the role of IgG anti-melanocyte antibodies in the inappropriate expression of major histocompatibility complex class II antigens and intercellular adhesion molecule-1 on melanocytes. IgG anti-melanocyte antibody samples were purified from the individual serum of patients with active vitiligo. After incubation of IgG anti-melanocyte antibodies with cultured melanocytes, the results revealed: (i) IgG anti-melanocyte antibody stimulated HLA-DR expression on melanocytes; (ii) intercellular adhesion molecule-1 expression on melanocytes was significantly induced by IgG anti-melanocyte antibodies; and (iii) IgG anti-melanocyte antibodies induced an increase in interleukin-8 release from melanocytes. The major histocompatibility complex class II molecules expressed in melanocytes can present antigens to CD4 helper cells as antigen-presenting cells and elicit an immune response. Intercellular adhesion molecule-1 is an important adhesion molecule involved in leukocyte and parenchymal cell interaction and thus plays an essential part in immunologic and inflammatory reactions. It is reasonable to speculate that abnormal expressions of HLA-DR and intercellular adhesion molecule-1 on melanocytes by IgG anti-melanocyte antibodies would present vitiligo antigens and allow the antigen-specific immune effector cell attack that results in melanocytotoxicity. mean fluorescence intensity IgG purified from normal control IgG purified from vitiligo patients Functional melanocytes in patients with vitiligo vulgaris disappear from involved skin by a mechanism(s) that has not yet been identified. An immunologic hypothesis is currently advanced as a possible pathogenesis of nonsegmental-type vitiligo. The presence of anti-melanocyte antibodies may play particularly important parts in the pathogenesis of vitiligo (Naughton et al., 1983aNaughton G.K. Eisinger M. Bystryn J.C. Antibodies to normal human melanocytes in vilitigo.J Exp Med. 1983; 158: 246-251Crossref PubMed Scopus (200) Google Scholar,Naughton et al., 1983bNaughton G.K. Esinger M. Bystryn J.C. Detection of antibodies to melanocytes in vitiligo by specific immunoprecipitation.J Invest Dermatol. 1983; 81: 540-542Crossref PubMed Scopus (127) Google Scholar). IgG anti-melanocyte antibodies (V-IgG) were found to induce melanocyte damage in vitro by a complement-mediated mechanism and antibody-dependent cellular cytotoxicity (Norris et al., 1988Norris D.A. Kissinger R.M. Naughton G.M. Bystryn J.C. Evidence for immunologic mechanisms in human vitiligo: Patients' sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody-dependent cellular cytotoxicity.J Invest Dermatol. 1988; 90: 738-789Google Scholar).al Badri et al., 1993al Badri A.M. Foulis A.K. Todd P.M. et al.Abnormal expression of, MHC, class, II, ICAM.-1 by melanocytes in vitiligo.J Pathol. 1993; 169: 203-206Crossref PubMed Scopus (83) Google Scholar reported an increase in major histocompatibility complex (MHC) class II antigens and intercellular adhesion molecule-1 (ICAM-1) expression in perilesional melanocytes in vitiligo; however, the mechanism for the abnormal expressions in vitiligo melanocytes has not yet been clarified. As these molecules possess important roles in cell–cell interactions and activation of helper T lymphocytes (Marlin and Springer, 1987Marlin S.D. Springer T.A. Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen-1 (LFA-1).Cell. 1987; 51: 813-819Abstract Full Text PDF PubMed Scopus (1413) Google Scholar;Dahl, 1996Dahl G. Where are the gates in gap junction channels?.Clin Exp Pharmacol P. 1996; 23: 1047-1502Crossref PubMed Scopus (23) Google Scholar), their expression on melanocytes may contribute to the abnormal immune response in vitiligo (al Badri et al., 1993al Badri A.M. Foulis A.K. Todd P.M. et al.Abnormal expression of, MHC, class, II, ICAM.-1 by melanocytes in vitiligo.J Pathol. 1993; 169: 203-206Crossref PubMed Scopus (83) Google Scholar). Identification of factors that induce MHC class II antigens and ICAM-1 expression in melanocytes may provide a crucial clue to the immunologic mechanisms of melanocyte destruction in patients with vitiligo. This study demonstrates that V-IgG elicits HLA-DR and ICAM-1 expression and an increase in interleukin (IL)-8 release by melanocytes. Twelve patients with vitiligo of nonsegmental-type were studied as in our previous report (Yu et al., 1997Yu H.S. Chang C.L. Yu C.L. Li H.F. Wu M.T. Wu C.S. Alterations in IL-6, IL-8, GM-CSF, TNF-α and IFN-γ release by peripheral mononuclear cells in patients with active vitiligo.J Invest Dermatol. 1997; 108: 527-529Crossref PubMed Scopus (90) Google Scholar). All patients had developed newly depigmented lesions in the previous 3 mo, i.e., they were suffering from active vitiligo (Moellmann et al., 1982Moellmann G. Klein-Angerer S. Scollay D.A. Nordlund J.J. Lerner A.B. Extracellular granular material and degeneration of keratinocytes in the normally pigmented epidermis of patients with vitiligo.J Invest Dermatol. 1982; 79: 321-330Crossref PubMed Scopus (180) Google Scholar). None of the patients had known autoimmune disorders. An equal number of age- and sex-matched healthy individuals served as controls. None of the participants had received medical treatment in the preceding 3 mo. The sera were collected from patients and normal controls for purification as in a previous study (Yu et al., 1989Yu C.L. Chang K.L. Hong S.T. Chiang B.N. Han S.H. Wang S.R. Defective helper T cell function in IgM rheumatoid factor synthesis in patients with ankylosing spondylitis.Scand J Rheumatol. 1989; 18: 43-49Crossref PubMed Scopus (6) Google Scholar). The fraction II (IgG) obtained from Sephacryl S-300 gel filtration was further absorbed by the protein A-Sepharose 4B (Pharmacia LKB, Uppsala, Sweden). Sepharose-conjugated IgG was eluted with sodium acetate buffer at pH 2.5 and immediately neutralized to pH 7.2 with 1 N NaOH. After adequate dialysis against distilled water, the IgG was lyophilized. The protein concentration of purified IgG was determined by Bio-Rad protein assay kit (Bio-Rad, Richmond, CA). These IgG preparations were found to contain negligible contaminants when detected by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar). The foreskins of adults from routine circumcisions were cleaned of excess subcutaneous tissue, cut into small pieces (5 × 5 mm), and incubated with 0.25% trypsin (Gibco, Santa Clara, CA) at 4°C overnight. The epidermal sheets were separated from dermis by fine forceps, and the epidermal cell suspension was prepared as reported previously (Kao and Yu, 1991Kao C.H. Yu H.S. A study of the effects of phorbol 12-myristate-13-acetate on cell differentiation of pure human melanocytes in vitro.Arch Dermatol Res. 1991; 283: 119-125Crossref PubMed Scopus (14) Google Scholar). The cells were pelleted by centrifugation (500 × g, 10 min) and resuspended in serum-free medium (Gibco). The epidermal cell suspension was plated on to plastic 10 cm2 Petri dishes, and incubated at 37°C in a humidified atmosphere containing 5% CO2. Twenty-four hours after primary seeding, the medium was changed, and then changed regularly every 2 d. About 7–10 d after primary seeding, the semiconfluent melanocytes were incubated with 0.25% trypsin in 0.01% ethylenediamine tetraacetic acid solution (Gibco) at 37°C for 3–5 min, harvested with fetal bovine serum (Gibco), centrifuged (500 × g, 10 min), and the cell pellet was resuspended in serum-free medium and re-incubated. The second-passage cells were used in the following experiments. Second-passage cells were used for V-IgG detection. The cells plated on coverslips were fixed with 100% methanol at -20°C for 3 min, incubated with purified IgG from the patients with active vitiligo [100 μg per ml in phosphate-buffered saline (PBS)] at room temperature for 60 min, then incubated with fluorescein isothiocyanate-conjugated rabbit anti-human IgG (DAKO, Glostrup, Denmark, 1:50 diluted in PBS) at room temperature for 60 min. Between each step, the cells were washed with PBS three times. The samples were observed using an Olympus BX-50 fluorescence microscope. The individual purified IgG was also measured for anti-melanocyte activity by cellular enzyme-linked immunosorbent assay (cellular ELISA) as reported previously (Yu et al., 1993Yu H.S. Kao C.H. Yu C.L. Coexistence and relationship of anti-keratinocyte and anti-melanocyte antibodies in patients with non-segmental-type vitiligo.J Invest Dermatol. 1993; 100: 823-828Abstract Full Text PDF PubMed Google Scholar). The melanocytes were subcultured into chamber slides (Nalge Nunc International, Naperville, IL) and incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. Cells were fixed in 100% ice-cold methanol for 3 min, washed three times with PBS, and incubated with 100 μg per ml V-IgG or normal IgG (N-IgG) at 37°C for 30 min. After washing with PBS three times, we added 100 μl of rabbit anti-human IgG (1:50 diluted) for 30 min at room temperature. Unbound rabbit anti-serum was washed three times with PBS, and a pig anti-rabbit peroxidase anti-peroxidase kit (Dako PAP Kit, K548) was used according to the manufacturer's instructions. Plates were read at 490 nm in an ELISA reader, and the relative titer of anti-melanocyte antibodies was expressed as optical density. Six samples containing the highest titer of V-IgG and six N-IgG samples with average titers were then individually used in the following experiments. The concentration of cytokines in the culture supernatants derived from 2 × 105 melanocytes per ml after a 24 h incubation with medium, V-IgG (100 μg per ml), N-IgG (100 μg per ml), or ultraviolet (UV) B irradiation at 100 mJ per cm2 (served as a positive control) was measured by commercially available ELISA kits including IL-8, IL-1α, IL-1β, and interferon-γ (IFN-γ) (Quantikine, R&D Systems, Minneapolis, MN). The results were presented as mean ± SD. The detectable concentration range was 31.2–2000 pg per ml for IL-8, 3.9–250 pg per ml for IL-1α, 3.9–250 pg per ml for IL-1β and 15.6–1000 pg per ml for IFN-γ. The same experiment was repeated nine times. Unpaired Student's t test was used to analyze the statistical differences between patients and controls. A difference of p <0.05 was considered as statistically significant. After an incubation of 72 h with either V-IgG (100 μg per ml) or N-IgG (100 μg per ml) in 3.5 cm culture dishes, melanocytes were detached with 0.25% trypsin in 1 mM ethylenediamine tetraacetic acid·4Na, washed twice with PBS buffer, pH 7.2, then incubated with fluorescein isothiocyanate-conjugated mouse anti-human ICAM-1(Leinco Technologies, Ballwin, MO), HLA-DP, HLA-DQ, or HLA-DR monoclonal antibody (Becton-Dickinson Immunocytometry Systems, Mountain View, CA) (1:20 diluted in Isoton solution) for 30 min in an ice-bath. Human recombinant tumor necrosis factor-α (TNF-α, 20 ng per ml) purchased from Quantikine, R&D Systems served as a positive control. The expressions of ICAM-1 and MHC Class II were detected by FACScan. Data were shown as mean fluorescence intensity (MFI, denoted by mean channel number) of ICAM-1 or MHC class II-positive cells. The same experiment was repeated nine times, and the statistical significance was analyzed by unpaired Student's t test. A difference of p <0.05 was considered as statistically significant. IL-8 mRNA expression in melanocytes after 6 h incubation with medium, V-IgG (100 μg per ml), or N-IgG (100 μg per ml) was measured by reverse transcriptase–PCR. One microgram of total RNA was reverse-transcribed into cDNA in the presence of 100 pmol poly deoxythymidine (Promega, Madison, WI), 1 mM deoxynucleoside triphosphate (Promega), avian myeloblastosis virus reverse transcriptase (4 units) (Promega), and 1 × reaction buffer (50 mM Tris (hydroxymethyl)–aminomethane–HCl, 50 mM KCl, 10 mM MgCl2,10 mM dithiothreitol, 0.5 mM spermidine) at 25°C for 10 min, 42°C for 40 min, 95°C for 10 min, and placed on ice. For the PCR reaction, we used the following primers: IL-8 5′-ACG- AATTCCTAGGACAAGCCAGGAAG-3′ (sense) and 5′-GTGAAT-TCAGTGTGGTCCACTCTCAATC-3′ (anti-sense) (Paludan and Thestrup-Pedersen, 1992Paludan K. Thestrup-Pedersen K. Use of the polymerase chain reaction in quantification of interleukin 8 mRNA in minute epidermis samples.J Invest Dermatol. 1992; 99: 830-835Abstract Full Text PDF PubMed Google Scholar), β-actin 5′-GTGGGGCGCCCCAGGCA- CCA-3′ (sense) and 5′-CTCCTTAATGTCACGCACGATTTC-3′ (anti-sense). Five microliter aliquots of synthesized cDNA were added to 95 μl of the PCR mixture containing 10 μl of 10 × PCR buffer (500 mM KCl, 100 mM Tris (hydroxymethyl)–aminomethane–HCl, 1% Triton X-100), 1 μl of sense and anti-sense primers (0.2 mM), 8 μl of 1.25 mM deoxynucleotide triphosphates, and 0.5 μl of Taq DNA polymerase (0.5 units per ml, Promega). The reaction mixture was overlaid with 80 μl of mineral oil. The mixture were then subjected to 35 cycles of 94°C for 1 min, 60°C for 1.5 min, and 74°C for 2 min after an initial denaturation step at 95°C for 5 min in a Hybaid OmniGene DNA Thermal Cycler (Teddington, Middlesex, TW, UK). The PCR products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. The DNA fragments amplified by these sets of primers were 253 bp for IL-8 and 548 bp for β-actin. Using the indirect immunofluorescence method, V-IgG showed a positive reaction with methanol-fixed melanocytes. Homogeneous fluorescence was found in the cytoplasm of the cells by V-IgG (Figure 1a), in contrast very faint staining was noticed in the cells by N-IgG (Figure 1b). By using methanol-fixed melanocytes as substrates in cellular ELISA, the optical density at 490 nm for a quantitative measure of anti-melanocyte activity of purified IgG, was significantly higher in V-IgG (0.70 ± 0.16) than N-IgG (0.25 ± 0.14) (p <0.05) (Yu et al., 1997Yu H.S. Chang C.L. Yu C.L. Li H.F. Wu M.T. Wu C.S. Alterations in IL-6, IL-8, GM-CSF, TNF-α and IFN-γ release by peripheral mononuclear cells in patients with active vitiligo.J Invest Dermatol. 1997; 108: 527-529Crossref PubMed Scopus (90) Google Scholar). The results indicated that there was a higher anti-melanocyte antibody titer present in V-IgG than that of N-IgG. In this study, we detected the expression of ICAM-1 and MHC class II (DR, DP, and DQ) on cultured melanocytes after incubation with either purified IgG from the patients with active vitiligo or normal IgG for nine experiments. The mean fluorescence intensity of ICAM-1, HLA-DR, -DP, and -DQ on V-IgG-, N-IgG-, TNF-α-stimulated and medium-incubated melanocytes is shown in Table 1. The results indicated that the expression of ICAM-1 and HLA-DR, but not HLA-DP and HLA-DQ, were significantly higher on cultured melanocytes incubated with V-IgG than incubated with N-IgG or medium (p <0.05, Table 1, Figure 2).Table IV-IgG induced a significant increase in HLA-DR and ICAM-1 expression on melanocytesaThe expression of MHC class II molecules (HLA-DR, -DP, and -DQ) and ICAM-1 on the cell surface of melanocytes was detected by flow cytometry after incubation with V-IgG, N-IgG, medium, and human recombinant TNF-α, as a positive control for 72 h. The same experiment was repeated nine times.Fluorescence intensitybThe fluorescence intensity of the cells was denoted by mean fluorescence intensity (mean channel number) ±SD. (% positive cells)Group n = 9HLA-DRHLA-DPHLA-DQICAM-1V-IgG (100 μg per ml)36.7 ± 6.4cp< 0.05, calculated by unpaired Student's t test when comparing V-IgG or TNF-α group to N-IgG group. (5.34)59.3 ± 20.741.2 ± 10.21268.7 ± 157.6cp< 0.05, calculated by unpaired Student's t test when comparing V-IgG or TNF-α group to N-IgG group. (35.41)N-IgG (100 μg per ml)20.7 ± 2.0 (0.85)50.8 ± 10.143.4 ± 4.6912.8 ± 178.2 (20.57)Medium22.2 ± 1.9 (0.64)48.5 ± 4.344.1 ± 13.1895.6 ± 102.2 (15.32)TNF-α (20 ng per ml)32.6 ± 5.2cp< 0.05, calculated by unpaired Student's t test when comparing V-IgG or TNF-α group to N-IgG group. (5.76)57.3 ± 7.345.8 ± 6.81375.7 ± 145.8cp< 0.05, calculated by unpaired Student's t test when comparing V-IgG or TNF-α group to N-IgG group. (32.14)a The expression of MHC class II molecules (HLA-DR, -DP, and -DQ) and ICAM-1 on the cell surface of melanocytes was detected by flow cytometry after incubation with V-IgG, N-IgG, medium, and human recombinant TNF-α, as a positive control for 72 h. The same experiment was repeated nine times.b The fluorescence intensity of the cells was denoted by mean fluorescence intensity (mean channel number) ±SD.c p< 0.05, calculated by unpaired Student's t test when comparing V-IgG or TNF-α group to N-IgG group. Open table in a new tab The release of IL-8 was significantly higher in cultured melanocytes incubated with V-IgG or UVB-irradiated cells as compared with that of N-IgG or unstimulated cells (p <0.001, Table 2) after nine experiments. In contrast, the release of IL-1α, IL-1β, and IFN-γ were not influenced by either V-IgG, N-IgG, or UVB stimulation (Table 2).Table IIV-IgG stimulated a significant increase in IL-8 release by melanocytesaThe effect of V-IgG, N-IgG, and UVB irradiation on the different cytokine production by human cultured melanocytes (2 × 105 per ml) after 24 h incubation detected by ELISA kit. The same experiment was repeated nine timesConcentration of cytokines (pg per ml, mean ± SD)Group n = 9IL-8IL-1αIL-1βIFN-γV-IgG (100 μg per ml)138.61 ± 24.22bp <0.001, calculated by unpaired Student's t test when comparing V-IgG or UVB treated group with N-IgG group.0.03 ± 0.070.11 ± 0.160.27 ± 0.10N-IgG (100 μg per ml)66.88 ± 8.180.04 ± 0.060.09 ± 0.080.20 ± 0.06Medium38.64 ± 4.280.03 ± 0.030.24 ± 0.120.14 ± 0.11UVB (100mJ per cm2)117.83 ± 15.67bp <0.001, calculated by unpaired Student's t test when comparing V-IgG or UVB treated group with N-IgG group.0.03 ± 0.020.21 ± 0.090.08 ± 0.05a The effect of V-IgG, N-IgG, and UVB irradiation on the different cytokine production by human cultured melanocytes (2 × 105 per ml) after 24 h incubation detected by ELISA kit. The same experiment was repeated nine timesb p <0.001, calculated by unpaired Student's t test when comparing V-IgG or UVB treated group with N-IgG group. Open table in a new tab IL-8 mRNA expression was detected by reverse transcriptase–PCR in melanocytes after incubation with V-IgG or N-IgG for 6 h (Figure 3). The cDNA was normalized to yield equivalent amounts of β-actin to standardize the amount of total cellular mRNA in each PCR reaction. The expression of IL-8 mRNA in IgG-stimulated melanocytes was higher than that of the unstimulated group. Furthermore, IL-8 mRNA expression in V-IgG-stimulated melanocytes was significantly higher than that of N-IgG-stimulated melanocytes. These results were compatible with the findings of IL-8 production measured by ELISA. Melanocytic cytotoxicity by immune mechanisms in vitiligo is a complex process involving effector–target attachment via pairs of adhesive molecules, activation of leukocyte receptors by target antigens and other surface markers, and triggering of cytolysis (Martz, 1987Martz E. LFA-1 and other accessory molecules functioning in adhesions of T and B lymphocytes.Hum Immunol. 1987; 18: 3-37Crossref PubMed Scopus (167) Google Scholar;Norris, 1990Norris D.A. Cytokine modulation of adhesion molecules in the regulation of immunologic cytotoxicity of epidermal targets.J Invest Dermatol. 1990; 95: 111S-120SAbstract Full Text PDF PubMed Google Scholar;Morelli and Norris, 1993Morelli J.G. Norris D.A. Influence of inflammatory mediators and cytokines on human melanocyte function.J Invest Dermatol. 1993; 100: 191S-195SAbstract Full Text PDF PubMed Google Scholar).Naughton et al., 1983aNaughton G.K. Eisinger M. Bystryn J.C. Antibodies to normal human melanocytes in vilitigo.J Exp Med. 1983; 158: 246-251Crossref PubMed Scopus (200) Google Scholar demonstrated that antibodies against melanocyte surface antigens exist in the serum of vitiligo patients. V-IgG were reported to induce melanocyte damage by a complement-mediated mechanism and antibody-dependent cellular cytotoxicity (Norris et al., 1988Norris D.A. Kissinger R.M. Naughton G.M. Bystryn J.C. Evidence for immunologic mechanisms in human vitiligo: Patients' sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody-dependent cellular cytotoxicity.J Invest Dermatol. 1988; 90: 738-789Google Scholar;Yu et al., 1993Yu H.S. Kao C.H. Yu C.L. Coexistence and relationship of anti-keratinocyte and anti-melanocyte antibodies in patients with non-segmental-type vitiligo.J Invest Dermatol. 1993; 100: 823-828Abstract Full Text PDF PubMed Google Scholar). V-IgG directed against 40–45 kDa or 75 kDa common-tissue antigens and a 65 kDa and 90 kDa pigment cell-specific antigen have also been reported by many authors (Cui et al., 1992Cui J. Harning R. Henn M. Bystryn J.C. Identification of pigment cell antigens defined by vitiligo antibodies.J Invest Dermatol. 1992; 98: 162-165Abstract Full Text PDF PubMed Scopus (102) Google Scholar,Cui et al., 1995Cui J. Arita Y. Bystryn J.C. Characterization of vitiligo antigens.Pigment Cell Res. 1995; 8: 53-59Crossref PubMed Scopus (53) Google Scholar;Kao and Yu, 1992Kao C.H. Yu H.S. Comparison of the effect of 8-methoxypsoralen (8-MOP) plus UVA (PUVA) on human melanocytes in vitiligo vulgaris and in vitro.J Invest Dermatol. 1992; 98: 734-740Abstract Full Text PDF PubMed Scopus (62) Google Scholar;Park et al., 1996Park Y.K. Kim N.S. Hann S.K. Im S. Identification of autoantibody to melanocytes and characterization of vitiligo antigen in vitiligo patients.J Dermatol Sci. 1996; 11: 111-120Abstract Full Text PDF PubMed Scopus (37) Google Scholar). The biopsy specimen of vitiligo lesions revealed an increase in the expression of MHC class II antigens and ICAM-1 on perilesional melanocytes (al Badri et al., 1993al Badri A.M. Foulis A.K. Todd P.M. et al.Abnormal expression of, MHC, class, II, ICAM.-1 by melanocytes in vitiligo.J Pathol. 1993; 169: 203-206Crossref PubMed Scopus (83) Google Scholar). Similar to type I diabetes, coexpression of MHC class II antigens and ICAM-1 render the cells immunogenic to potentially auto-reactive T lymphocytes (Campbell and Harrison, 1990Campbell I.L. Harrison L.C. A new view of the beta cell as an antigen-presenting cell and immunogenic target.J Autoimmun. 1990; 3: 53-62Crossref PubMed Scopus (14) Google Scholar. In this study, three interesting findings were observed: (i) V-IgG stimulated HLA-DR expression on melanocytes; (ii) V-IgG stimulated ICAM-1 expression on melanocytes; and (iii) V-IgG induced an increase in IL-8 release from melanocytes. MHC class II molecules can present exogenous antigens to CD4 helper cells and are involved in certain cell–cell interaction and augment immune responses (Dahl, 1996Dahl G. Where are the gates in gap junction channels?.Clin Exp Pharmacol P. 1996; 23: 1047-1502Crossref PubMed Scopus (23) Google Scholar). Although no experiment was conducted in this aspect, we speculate that the increased HLA-DR expression on melanocytes may enhance the antigen-presenting activity on the cells. In addition, increased expression of MHC class II antigens per se may trigger lymphocyte reactions including cytotoxic T lymphocytes (Campbell and Harrison, 1990Campbell I.L. Harrison L.C. A new view of the beta cell as an antigen-presenting cell and immunogenic target.J Autoimmun. 1990; 3: 53-62Crossref PubMed Scopus (14) Google Scholar), which may cause melanocytolysis. ICAM-1, as a member of the integrin family, is involved in cell–cell interactions of leukocytes and parenchymal cells, and thus plays an important part in the induction of immunologic and inflammatory reactions. The ligand for ICAM-1 is leukocyte function associated antigen-1 which is expressed by all leukocytes (Martz, 1987Martz E. LFA-1 and other accessory molecules functioning in adhesions of T and B lymphocytes.Hum Immunol. 1987; 18: 3-37Crossref PubMed Scopus (167) Google Scholar). Leukocyte function associated antigen-1/ICAM-1 interactions have been shown to be important for controlling cytotoxicity mediated by lymphokine-activated killer cells (Kalish, 1989Kalish R.S. Non-specifically activated human peripheral blood mononuclear cells are cytotoxic for human keratinocytes in vitro.J Immunol. 1989; 142: 74-80PubMed Google Scholar), cytotoxic lymphocytes (Shau et al., 1986Shau H.Y. Shen D. Golub S.H. The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function.Cell Immunol. 1986; 97: 121-130Crossref PubMed Scopus (18) Google Scholar), and antibody-dependent cellular cytotoxicity effector cells (Miedema et al., 1984Miedema F. Tetteroo P.A. Hesselink W.G. Werner G. Spits H. Melief C.J. Both Fc receptors and lymphocyte-function-associated antigen 1 on human T gamma lymphocytes are required for antibody-dependent cellular cytotoxicity (killer cell activity).Eur J Immunol. 1984; 14: 518-523Crossref PubMed Scopus (83) Google Scholar). Once melanocytes express an excess of adhesion molecules an increased susceptibility to immune cytotoxic damage occurs.al Badri et al., 1993al Badri A.M. Foulis A.K. Todd P.M. et al.Abnormal expression of, MHC, class, II, ICAM.-1 by melanocytes in vitiligo.J Pathol. 1993; 169: 203-206Crossref PubMed Scopus (83) Google Scholar reported a significant increase in the expression of MHC class II and ICAM-1 on perilesional melanocytes of vitiligo specimens. MHC class II and ICAM-1 play important parts in normal antigen presentation and activation of helper T lymphocytes, and their expression by melanocytes may contribute to the abnormal immune response in vitiligo. In this study, V-IgG induced a significant increase in the expression of HLA-DR and ICAM-1 of melanocytes (Table 1). The result is consistent with the report presented byal Badri et al., 1993al Badri A.M. Foulis A.K. Todd P.M. et al.Abnormal expression of, MHC, class, II, ICAM.-1 by melanocytes in vitiligo.J Pathol. 1993; 169: 203-206Crossref PubMed Scopus (83) Google Scholar. Elevated expression of ICAM-1 and MHC class II in association with certain putative vitiligo antigens would allow for antigen-specific immune-effector-cell attachment and cause melanocyte cytotoxicity. Furthermore, in individuals with circulating antibodies against vitiligo antigens, the serum may enhance the antibody-dependent cellular cytotoxicity killing mechanism on those melanocytes with increased melanocyte ICAM-1 expression (Yohn et al., 1990Yohn J.J. Critelli M. Lyons M.B. Norris D.A. Modulation of melanocyte intercellular adhesion molecule-1 by immune cytokines.J Invest Dermatol. 1990; 90: 233-237Crossref Scopus (72) Google Scholar). The reason for only a subpopulation of cells responding with increased MHC class II and ICAM-1 expression on melanocytes in vitiligo specimens and in V-IgG stimulation experiments is not known. Two possibilities could be responsible: (i) the density of HLA-DR and ICAM-1 molecules on the surface of either cultured melanocytes or biopsied tissues is not enough to be detected by the methods used in all cells, or (ii) the melanocytes in culture or in situ are not in the same stage of the cell cycle, and therefore differentially susceptible for HLA-DR or ICAM-1 induction. Some studies demonstrated that inflammatory reactions occur at the margin of vitiligo, i.e., accumulation of lymphocytes and activated T lymphocytes near surviving melanocytes (al Badri et al., 1993al Badri A.M. Foulis A.K. Todd P.M. et al.Abnormal expression of, MHC, class, II, ICAM.-1 by melanocytes in vitiligo.J Pathol. 1993; 169: 203-206Crossref PubMed Scopus (83) Google Scholar;Galadari et al., 1993Galadari E. Mehregan A.H. Hashimoto K. Ultrastructural study of vitiligo.Int J Dermatol. 1993; 32: 269-271PubMed Google Scholar;MoSeries et al., 1993MoSeries B. Barella L. Mattei S. Schumacher C. Boulay F. Colombo M.P. Baggiolini M. Expression of transcripts for two interleukin 8 receptors in human phagocytes, lymphocytes and melanoma cells.Biochem J. 1993; 294: 285-292Crossref PubMed Scopus (115) Google Scholar). In this study, we confirmed IL-8 stimulatory effects of V-IgG on melanocytes, either by ELISA or reverse transcriptase–PCR. In addition to melanocytes, V-IgG induced an increase in IL-8 release from cultured keratinocytes (data not shown). It is conceivable that IL-8 is a chemoattractant/activator for T lymphocytes (Taub et al., 1996Taub D.D. Anver M. Oppenheim J.J. Longo D.L. Murphy W.J. T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.J Clin Invest. 1996; 97: 1931-1941Crossref PubMed Scopus (243) Google Scholar) as well as for polymorphonuclear leukocytes. The recruitment of T lymphocytes to the site of vitiligo may be partly due to the attraction of IL-8 release from both melanocytes and keratinocytes stimulated by V-IgG; however, the cause of scanty polymorphonuclear leukocyte infiltration in the vitiligo lesion is not clear at the present time. A previous study demonstrated a significant increase in spontaneous production of IL-6 and IL-8 by mononuclear cells of patients with active vitiligo. V-IgG stimulates an increase in IL-6 production by peripheral mononuclear cells (Yu et al., 1997Yu H.S. Chang C.L. Yu C.L. Li H.F. Wu M.T. Wu C.S. Alterations in IL-6, IL-8, GM-CSF, TNF-α and IFN-γ release by peripheral mononuclear cells in patients with active vitiligo.J Invest Dermatol. 1997; 108: 527-529Crossref PubMed Scopus (90) Google Scholar). It is reported that IL-6 can induce the expression of cell surface ICAM-1 on melanocytes (Kirnbauer et al., 1992Kirnbauer R. Charvat B. Schauer E. et al.Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: Evidence for a regulatory role of IL-6, IL-7, TNF-β and UVB light.J Invest Dermatol. 1992; 98: 320-326Abstract Full Text PDF PubMed Scopus (78) Google Scholar). IL-6 secreted by T lymphocytes and monocytes/macrophages in inflammatory reaction at the margin of vitiliginous lesions may accentuate ICAM-1 expression on melanocytes in addition to V-IgG. Increased IL-8 production from melanocytes by V-IgG may specifically chemo- attract T lymphocytes to the site of lesions to amplify the inflammatory reaction and facilitate melanocytic cytotoxicity. In conclusion, our results are consistent with the possibility that V-IgG play an important role in melanocytic cytotoxicity immune-mediated: (i) increased HLA-DR expression on melanocytes enhances the antigen-presenting activity of the cells; (ii) ICAM-1 expression on melanocytes stimulates leukocyte/monocyte attachment and immune-mediated cytotoxicity; and (iii) increased IL-8 production from melanocytes chemoattracts T lymphocytes to the sites of lesions to increase melanocyte destruction. This study was supported by the National Science Council of the Republic of China (Taiwan) Research grant NSC 85–2331-B-037–088.