Background/Purpose:

Since their detection a pathophysiological role of antibodies (abs) to extractable nuclear autoantigens including anti-La abs has been controversially discussed. Stainings of anti-La abs point to an exclusive nuclear localization. However, we and others have also seen an intracellular shuttling and an occurrence on the cell surface. All conditions leading to a translocation of La onto the cell surface were later found to induce apoptosis. Therefore, a pathophysiological contribution of anti-La abs appears rather unlikely. And, even until today, the mechanisms underlying the shuttling and translocation of the autoantigen are still largely unclear.

Methods:

Ten novel monoclonal abs (mabs) were established, characterized by epitope mapping and used for analysis of intra- and cell surface localization of La by immunoblotting, ELISA, epifluorescence microscopy and FACS. The observed redox dependent localization was confirmed by analysis of native and cystein mutant (GFP) La forms. In addition to mabs, human anti-La abs directed to redox dependent forms of La were isolated from autoimmune sera, tested for binding to cell surface La and induction of an ADCC using Chromium-release and FACS-based cytotoxicity assays.   

Results:

Reactivities of novel anti-La mabs were found to depend on reducing/non-reducing conditions when analyzed by immunoblotting or ELISA. The sensitivity to oxidation was lost when all three cystein residues present in La were mutated. Oxidation of native La protein or cystein mutants in which only one or two cystein residues were mutated leads to inter- and intra cystein disulfide bonds, and thereby to the formation of dimers and higher oligomers in vitro and in vivo. Oxidative stress results in  oxidation of La and causes a separation from a redox dependent nuclear retention partner. Thereafter, La translocates to the cytoplasm and its reentry into the nucleus is blocked resulting in a cytoplasmic enrichment. Oxidation of La can occur in a cell type specific manner. E.g. LPS treatment causes a translocation only in cells expressing TLR4. Dying cells can release La. Released La binds strongly to the surface of neighbouring intact cells. The binding depends on the cell type and the oxidative status of the protein. Only oxidized La binds strongly to the surface of cells including to epithelial, endothelial, blood DCs and other APCs but not to NK- or T cells. Surface bound La is available for binding of anti-La abs, thereby mediating ADCC. Furthermore, it can bind nucleic acids including DNA which improves the binding of other autoantibodies such as anti-DNA abs. Such La/nucleic acid complexes can lead to maturation of DCs.

Conclusion:

Under physiological conditions the shuttling of La is favoured to a nuclear localization. Redox dependent conformational changes of La alter this balance resulting in an enrichment of La in the cytoplasm. Dead cells release La which then binds to the surface of intact living cells in a redox dependent manner. Anti-La abs can bind to surface La and cause an ADCC. These redox dependent alterations explain why anti-La abs can become of pathophysiological relevance only under conditions causing acute phases of disease in autoimmune patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/redox-dependent-conformational-changes-of-the-autoantigen-la-are-responsible-for-its-shuttling-translocation-and-a-pathophysiological-role-of-anti-la-autoantibodies/