RESUMO
Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.
Assuntos
Anticorpos Antinucleares/química , Anticorpos Antifosfolipídeos/química , Apoptose/imunologia , Glicoproteínas/imunologia , Fragmentos de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Fosfatidilserinas/imunologia , Sequência de Aminoácidos , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/imunologia , Anticorpos Antifosfolipídeos/genética , Anticorpos Antifosfolipídeos/imunologia , DNA/imunologia , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Células Jurkat , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , beta 2-Glicoproteína IRESUMO
The target of many antiphospholipid autoantibodies (APA) has been shown to be a complex between anionic phospholipid (PL) and the plasma protein beta 2-glycoprotein I (beta 2-GPI), but the identity of the natural target(s) and/or immunogen for APA in vivo remains undetermined. The anionic PL of cell membranes represent important potential targets and immunogenes for APA. Although anionic PL are normally absent from the extracellular surface of cell membranes, they redistribute from the inner to the outer leaflet during apoptosis. We and others have shown that beta 2-GPI binds selectively to the surface of apoptotic, but not viable, cells, and that the binding of beta 2-GPI to the surface of apoptotic cells generates an epitope recognized by APA from patients with both primary antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). In this review, we discuss recent findings, which suggest not only that apoptotic cell-bound beta 2-GPI is injected by non-intravenous routes. We also review briefly the potential role of oxidation in generating epitopes responsible for the recognition and induction of APA. Taken together, we believe that the available evidence supports a role for apoptotic cells as far as targets of APA and possible players in the induction of APA.
