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Vitiligo
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In situ immune infiltrates in lesional, perilesional, and nonlesional skin biopsies from patients with vitiligo were analyzed by immunohistochemistry and compared with immune infiltrates found in the skin of normal healthy donors and relevant disease controls. An increased influx of activated skin-homing T cells and macrophages were seen in the perilesional biopsies. The overall percentages of cutaneous leukocyte-associated antigen-positive (CLA+) T cells were similar to those found in normal healthy donors. This is compatible with the similar expression of E-selectin. Most strikingly, however, the CLA+ T cells in perilesional skin were mainly clustered in the vicinity of disappearing melanocytes, and 60% to 66% of these interacting T cells expressed perforin and granzyme-B. The perforin+/granzyme-B+ cells were not seen in locations different from that of disappearing melanocytes. Interestingly, the majority of the infiltrating T cells were HLA-DR/CD8+. Another hallmark of the present study is the focal expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR in the epidermis at the site of interaction between the immune infiltrates and the disappearing melanocytes. The data presented in this study are consistent with a major role for skin-homing T cells in the death of melanocytes seen in vitiligo.
Patients with vitiligo often have antibodies to pigment cells. To examine whether there is a relation between the presence of such antibodies and disease activity, sera of 24 patients with vitiligo (10 with active and 14 with inactive disease) and 19 normal individuals were tested for antibodies to pigment cell surface antigens using a live cell enzyme-linked immunoabsorbent assay. IgG pigment cell antibodies were present in 80% (eight of 10) of patients with active vitiligo but in none of those with inactive disease or in normal individuals. The antibody level of patients with active vitiligo (mean binding index [BI] 3.3 +/- 0.59) was significantly higher than in patients with inactive disease (BI 0.96 +/- 0.04) or normal individuals (BI 1.0 +/- 0.04, p less than 0.001). Antibodies present in eight patients with high titers of pigment cell antibodies reacted to three of four pigment cells but to only one of six unrelated cells. These findings indicate that a correlation exists between the incidence and level of pigment cell antibodies and the activity of vitiligo, and support the hypothesis that vitiligo is an autoimmune disease mediated by an immune reaction to pigment cells.
Patients with vitiligo have circulating antibodies to melanocytes. To identify vitiligo antibodies and characterize the antigens by vitiligo antibodies, sera of 18 patients with vitiligo, 18 with Behcet's disease, 22 with syphilis and 14 normal control subjects were analyzed by indirect immunofluorescence, live cell ELISA, and immunoblotting. In indirect immunofluorescent microscopy and live cell ELISA, most vitiligo sera showed positive immunofluorescence and high optical density on the surface of melanocytes cultured from normal and vitiligo patients, indicating that autoantibodies in the vitiligo sera may react with vitiligo antigens on the surface of melanocytes. When the same experiments were performed with malignant melanoma cell lines and fibroblasts, no significant differences in the immunofluorescence and optical density were observed between normal and vitiligo sera. And the sera of patients with Behcet's disease or syphilis showed no significant difference in the reaction of live cell ELISA to fibroblasts, IGR-3 and melanocytes. The antibody titers of vitiligo patients in live cell ELISA decreased following systemic steroid treatments. Immunoblot analysis demonstrated that 44% of vitiligo sera was directed to melanocyte antigen with a molecular weight of 65 kDa. Inhibition assay using rabbit anti-melanocyte antibody showed inhibition of reaction between vitiligo sera and melanocytes in ELISA and immunoblotting. These findings support the hypothesis that the sera of vitiligo patients have autoantibodies mostly directed to the 65-kDa antigen and this antigen may originate mostly from the melanocyte surface.
In our previous studies, we found that polyclonal anti-double-stranded DNA antibodies (anti-dsDNA) purified from sera of patients with active systemic lupus erythematosus (SLE) were cytotoxic to cultured rat glomerular mesangial cells (RMC) through an apoptotic mechanism. In order to determine whether these nephritogenic antibodies affect the expression of apoptosis-related genes in the tissues, the expression of Fas, p53, c-myc, and bcl-2 genes in the kidneys and livers of 12-week-old normal BALB/c and autoimmune MRL-lpr/lpr mice was detected by a reverse transcription-assisted polymerase chain reaction (RT-PCR). We found the mRNA of the four genes were expressed in the tissues of the normal mice. In contrast, decreased expression of the four genes in the kidney and absent expression of bcl-2 in the liver of the lpr mice were noted. Interestingly, RMC only expressed p53 and c-myc, but not Fas or bcl-2, in culture. The purified polyclonal anti-dsDNA dose-responsively (50-200 IU/ml) suppressed the 3H-thymidine incorporation of RMC after incubation for 48 h. However, the incubation of 100 IU/ml of anti-dsDNA with RMC for 4 h did not affect the expression of these apoptotic genes. The results suggest that anti-dsDNA induce RMC apoptosis via an unidentified mechanism different from Fas, c-myc or p53 pathway.
IT is commonly accepted that antibodies do not penetrate living cells. In only one study anti-purine and anti-nucleoside antibodies were found to penetrate fertilised sea urchin eggs and modify their development1. Such penetration has been considered unusual and the addition of anti-DNA antibodies does not affect mammalian tissue cells in culture2. Direct immunofluorescence of skin biopsies of patients with mixed connective tissue disease (MCTD) using fluorescent anti-IgG has occasionally shown speckled intranuclear fluorescence3-5 but it is doubted that IgG entered the cells while still viable. Patients with MCTD have high titres of antibody to nuclear ribonucleoprotein (RNP)6,7 which also gives a nuclear speckled pattern on cell substrates in direct immunofluorescence8. Should the antibodies to cellular components and nucleic acids which occur in autoimmune diseases be able to penetrate living cells, a novel mechanism of immunologically mediated damage and/or dysfunction could operate. We show here that anti-RNP IgG can penetrate viable human mononuclear cells (MNC), by their surface Fc receptor, and react with their nuclear RNP.
In view of evidence suggesting vitiligo is an autoimmune disease, we investigated whether vitiligo is associated with inherited deficiencies of the fourth (C4) and second (C2) component of complement and with certain human leukocyte antigens (HLA). Analysis of functional activities of C4 and C2 in sera of patients with vitiligo (n = 42) showed that 17% of them had a heterozygous C4 deficiency and 5% had a heterozygous C2 deficiency. In the normal control group (n = 30), 3% had a heterozygous C4 deficiency and none had a C2 deficiency. C4 typing by Western blot analysis showed the frequency of the C4A*Q0 allele in the vitiligo patient group to be close to normal. However, the frequency of one C4B*Q0 allele was three times higher, and that of two C4B*Q0 alleles five times higher in the vitiligo patient group than the reported frequencies in normal control groups. Southern blot analysis of Taq1 digests of DNA using C4 and 21-hydroxylase probes showed that two patients with two C4B*Q0 alleles had a deletion of a 21-OHA-C4B segment. In the other patients, having one or two C4B*Q0 alleles, these null alleles probably occurred due to a loss of C4 gene expression. HLA analysis did not show any allelic association of C4A*Q0 or C4B*Q0 with any HLA antigen in vitiligo, but confirmed the previous findings of a negative association with HLA-DR3 and a positive association with HLA-DR4. These results suggest that abnormalities of the C4B gene and the above-mentioned associations with HLA antigens may be some of the risk factors in vitiligo.
We identified the ADP/ATP carrier, located within the inner mitochondrial membrane, to be an organ- and conformation-specific autoantigen in myocarditis and dilated cardiomyopathy. We also showed that autoantibodies to the ADP/ATP carrier inhibit the nucleotide transport in vitro. Specific binding of the autoantibodies to the carrier was demonstrated by radioimmunoassay and the immunoblot technique; the inhibition of the nucleotide transport was determined by the inhibitor stop method. To establish if these autoantibodies might also affect cardiac energy metabolism in vivo, we measured whether they are capable of penetrating into myocytes and whether subcellular ATP/ADP ratios and phosphorylation potentials of ATP change in hearts of guinea pigs that have been immunized with the isolated ADP/ATP carrier. An intracellular deposition of autoantibodies was observed by direct immunofluorescence and by immunoperoxidase staining on cryosections of the myocardial tissue of animals immunized with the ADP/ATP carrier. Furthermore, binding of autoantibodies to mitochondrial membrane structures was shown by immunoelectron-microscopic methods. The cytosolic and intramitochondrial distribution of adenine nucleotides in stimulated, isolated perfused hearts of guinea pigs immunized with the ADP/ATP carrier was measured by nonaqueous fractionation. Compared with controls performing equal external heart work, the cytosolic ATP decreased in the immunized animals, whereas the mitochondrial ATP increased strongly; ADP concentrations showed an opposite change. Thus, a resultant cytosolic decrease and a marked mitochondrial increase of the ATP/ADP ratio was established. As a consequence, the cytosolic-mitochondrial phosphorylation potential of ATP was diminished. These findings demonstrate that antibodies against intracellular antigens are able to penetrate into living cells, and that autoimmunity to the ADP/ATP carrier may contribute to the pathophysiology of myocarditis and dilated cardiomyopathy by causing an autoantibody-mediated imbalance between intracellular energy delivery and demand.
Vitiligo is an acquired depigmentary disease of the skin that is caused by a selective destruction of melanocytes in discrete, usually symmetric, patches on the skin. This results in sharply outlined areas of complete depigmentation, which are particularly prominent in individuals with dark skin. Vitiligo is common, involving approximately 1% of the world's population. Although it is asymptomatic and does not adversely affect survival, the disease can be disfiguring and cause severe psychologic stresses and difficulties with employment. The cause of vitiligo is not known; however, a striking aspect of the disease, which may be providing a clue to its pathogenesis, is that the damage it causes is highly selective, being almost entirely restricted to melanocytes.
Human vitiligo is a common depigmenting disorder that is often associated with polyendocrinopathies. The etiology of vitiligo is unknown although there is indirect evidence of a strong association between antimelanocyte antibodies in animal and human vitiligo. We report direct evidence that vitiligo patients' sera containing antimelanocyte antibodies can lyse cultured human melanocytes by both complement activation and antibody-dependent cellular cytotoxicity (ADCC). Melanocyte cytotoxicity was measured using an ethidium bromide/acridine orange viability assay, after 4 and 16 h incubation with sera from vitiligo patients and from normal controls. Significant melanocyte cytotoxicity was seen with vitiligo patients' sera as an antibody source with both complement-mediated cytotoxicity (p less than 0.01) and ADCC (p less than 0.05) as effector mechanisms. Nine of 11 vitiligo patients' sera produced cytotoxicity by complement-mediated lysis or ADCC. No cytotoxicity was seen using fibroblast targets and vitiligo patients' sera. The lysis of human melanocytes by vitiligo patients' sera by two different effector mechanisms provides direct support for the autoimmune hypothesis of human vitiligo.
Microsome, thyroid and DNA tests on patients with generalized vitiligo vulgaris, with or without Sutton's leukoderma, show a high positive rate without thyroid diseases. A small, but not negligible, amount of IgG and C3 deposits in the basement-membrane zone and keratinocytes, has been frequently seen in these diseases by immunohistology and immuno-electron microscopy. This evidence strongly suggests an autoimmune character for these diseases.