| BackgroundVitiligo is a common disorder of skin depigmentation characterized by the destruction of melanocytes and its pathogenesis is still unknown. Recently, it has been demonstrated that the apoptosis induced by oxidative stress might play an initial role in melanocyte destruction. The proteins of apoptotic melanocyte with immunogenicity can induce autoimmunity, causing immune destruction of melanocytes and finally leading to the development of vitiligo. Furthermore, the discovery of the cytotoxic T lymphocytes(CTLs) infiltrating around the skin lesion that can kill melanocytes as well as the antibody specific for melanocytes in the blood of vitiligo patients indicate the main role of cellular and humoral immunity in the destruction of melanocyte. Previous studies have identified some related antigens specific for the immune response in vitiligo, but the molecular mechanism of the exposure, the recognition and the phagocytosis of intracellular antigens in melanocytes still unknown.It has been reported that apoptotic bodies(ABs) that have received more attention in recent years possess autoimmune-related proteins including heat shock protein, histone, enolase and so on. Research on systemic lupus erythematosus(SLE) has demonstrated that autoantigens accumulate to the cell surface during cell apoptosis, and the ingredients of antigens relocate to the ABs through cell blebbing. Then, ABs can be phagocyted by the surrounding phagocytes or non-professional APCs, thus forming the immune presentation of autoantigens and inducing immune response. Study on the mesenchymal stem cell deriving from insulinoma showed that autoantigens and immune adjuvants in the extracellular vesicles could induce T cell response. In addition, Buzas et al. found that cells can release ABs that contain damage associated molecular patterns(DAMPs), thus inducing and maintaining the inflammation under the condition of stress and damage. These findings indicate that ABs can act as the carrier of autoantigens and danger signals, playing a key role in autoimmune diseases. These discoveries bring us some new thoughts and exploration directions, that is, whether melanocytes can produce ABs under the oxidative stress, whether these ABs contain autoantigens and play a role in the initiation and development of vitiligo, and whether ABs can bridge apoptosis induced by oxidative stress and immune destruction of melanocyte?Based on the literature and the previous work of our team, we hypothesized that oxidative stress initiates the autoantigens to form the ABs, prompts the recognition and phagocytosis of ABs by the activated epidermal langerhans cells(LCs) through the activation of apoptosis-related signaling pathways like JNK and caspase, and finally induces the specific immune response, promoting the development of vitiligo. Methods:The immortalized melanocytes PIG3 V from patients with vitiligo were involved in this research. Apoptosis was induced by oxidative stress. ABs were isolated and purified and observed by transmission electron microscope. The quality of ABs was further tested by the detection of phosphatidylserine(PS) on the surface of particles using flow cytometry. The autoantigens in ABs were identified by Western Blot and verified by proteomics. The colocalization of antigen and ABs were detected by immunoelectron microscopy. The morphologic changes of ABs and the translocation of antigens under different condition of oxidative stress were observed by transmission electron microscope and Western Blot, and the formation of ABs was analyzed by flow cytometry(FSC/SSC analysis). Whether there is a positive relation between the expression of these antigens, the formation of ABs and the time and dose of oxidative stress was analyzed. The relevance between autoantigens, the formation of ABs and oxidative stress were also discussed. Then PIG3 V cells were pretreated with inhibitors of JNK, caspase, and cytoskeletal activation before induced apoptosis by oxidative stress, and the formation of ABs and the translocation of antigens in ABs were detected through flow cytometry and Western Blot. The influence of inhibitors of these pathways on the formation of ABs and translocation of autoantigens were identified. Results:1. Apoptosis was assessed in PIG3 V cells. Compared with the control cells, the number of apoptotic and necrotic cells increased in a dose-dependent manner after H2O2 stimulation over a 24-hour period. From these data, specific concentrations of H2O2 were chosen according to the induction of a statically significant increase in apoptosis but not necrosis(800μmol/l).2. The vesicles were isolated and purified by the combination of differential centrifugation and filtration of the supernatant of PIG3 V cell cultures. The morphology of the samples was determined by transmission electron microscopy. Further more, the PS on them was identified by flow cytometry, more than 90% of particles were Annexin V+. Together, these data demonstrated that ABs were separated successfully.3. We performed Western Blot to test some vitiligo-related antigens we had known, including TYR, TYRP-1, and TYRP-2 as well as Lamin A and Lamin A(ASP230) that we discovered before, and elucidate that Lamin A(ASP230) and TYRP-1 can relocate to ABs during apoptosis. Meanwhile, we found that some other antigens in the cytoplasm and nucleus could relocate to ABs during the process of apoptosis. We further testified these results by proteomics. In addition, we observed the colocalization of these antigens and ABs by immunoelectron microscopy.4. The morphology variation of ABs from PIG3 V stimulated by different strength and time course was observed by transmission electron microscopy. There was no obvious change with the increasing concentration, but more fragments of cells emerged with ABs when we used the concentration which could induce necrosis. In addition, with the time course extended, the ABs gradually swelled and shattered.5. The variation of antigens translocated in ABs from melanocyte stimulated by different strength and time course was observed by Western Blots. We found that antigens in ABs have a positive correlation with the strength and time of oxidative stress stimulation. Further more, flow cytometry analysis proved that the formation of ABs had a positive correlation with the strength and time of oxidative stress.6. We pretreated cells with the inhibitors of JNK(SP600125), caspase(Z-VAD), and cytoskeletal activation(Y-27632 and ML-9) and then to induce apoptosis. We found that the translocation of autoantigens in ABs was inhibited by blocking these pathways, respectively. Further more, flow cytometry analysis proved that the inhibition of these pathways could inhibit the formation of ABs. The use of these inhibitors revealed that translocation of autoantigens and formation of ABs depends on the activation of the apoptotic signaling cascade(JNK and caspase activation) and the consecutive cytoskeletal activation. Conclusion:In the present study, we characterized ABs isolated from apoptozing PIG3 V. We found an accumulation of some vitiligo-related antigens like Lamin A(ASP230) as well as TYRP-1 in ABs. Further more, we found that pathways including JNK, caspase, and the consecutive cytoskeletal activation play a very important role in in the formation of ABs and the translocation of autoantigens in ABs. Combined with recently reported data, our research demonstrates that oxidative stress can initiate caspase cascade and induce apoptosis through the activation of JNK signal pathway. Meanwhile, vitiligo-related autoantigens take part in the formation of ABs. Moreover, with the concentration increasing and the time course extending, the integrity of AB membrane is damaged. This result indicats that ABs may be the way by which the intracellular antigens releasing directly. In summary, this research demonstrates that the autoantigens of melanocyte can deform and accumulate in the ABs after apoptosis under oxidative stress. It’s probably also a way for the intracellular antigens exposure and results in the clone of specific CTLs and the activation of autoreactive B cells. Our study provides a new proof of the mechanism of how oxidative stress activates autoimmune response and further enrich the pathogenesis of vitiligo. |
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