Background/Purpose: β2-glycoprotein I (β2GPI), a phospholipid-binding protein, binds to apoptotic cells and serves as an antigenic target for autoantibodies from patients with systemic lupus erythematosus (SLE). Here, we determine whether this paradigm can be extended to necroptotic cells. Like apoptotic cells, necroptotic cells express phosphatidylserine on their surface and provide a “scaffold” of cellular self-antigens. We hypothesized that β2GPI should bind to necroptotic cells and serve as a target for anti-β2GPI autoantibodies from patients with SLE.

Methods: We established conditions for inducing necroptotic or apoptotic cell death in murine L929 fibroblast cells. L929 cells treated with vehicle served as the viable cell control. Cells were incubated with human β2GPI and murine monoclonal anti-β2GPI IgG antibody (or isotype control), and bound antibody detected with fluorescently conjugated anti-murine IgG. Similarly, binding of anti-β2GPI autoantibodies from human sera (or IgG isolated from these sera) was detected using fluorescently conjugated anti-human IgG. Human sera were from healthy controls and patients who satisfied the ACR classification criteria for SLE. Antibody binding was analyzed by flow cytometry and confocal microscopy.

Results: Using murine monoclonal anti-β2GPI antibody, we demonstrate that β2GPI binds to necroptotic cells in a similar manner as to apoptotic cells, but not to viable cells. Cells treated with an irrelevant antigen (serum albumin) or isotype control showed little or no antibody binding. Confocal microscopy confirmed monoclonal anti-β2GPI antibody staining of the cell surface of both necroptotic and apoptotic cells. SLE sera positive for anti-β2GPI autoantibodies bound to necroptotic cells and apoptotic cells. In contrast, sera negative for anti-β2GPI autoantibodies behaved similarly to healthy control sera, and showed little or no binding to necroptotic or apoptotic cells. Binding of purified IgG from the sera replicated the findings observed with sera.

Conclusion: Our data demonstrate that β2GPI binds to necroptotic cells and can serve as a target for SLE autoantibodies recognizing β2GPI. Based on these findings, we propose that the paradigm of apoptotic cells serving as a “cellular scaffold” of self-antigens in SLE may be extended to necroptotic cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/%ce%b22-glycoprotein-i-binds-to-necroptotic-cells-and-serves-as-a-target-for-sle-autoantibodies/