Antiphospholipid Antibodies Stimulate Release Of Endothelial Cell Microparticles Enriched In IL-1β

Microparticles are submicron vesicles released by activated or apoptotic cells. Previous studies from our laboratory and others have demonstrated elevated levels of endothelial cell (EC)-derived microparticles in the plasma of patients with antiphospholipid antibodies (APLA), which are associated with thrombosis and recurrent fetal loss. The majority of pathologic APLA are directed against β₂-glycoprotein I (β₂GPI), an abundant plasma phospholipid binding protein. APLA/anti-β₂GPI antibodies activate endothelial cells in a β₂GPI-dependent manner and stimulate the release of EC microparticles. Here, we demonstrate that these microparticles are enriched in active interleukin-1β (IL-1β) and may directly stimulate EC activation.

EC were treated with β₂GPI and anti-β₂GPI antibodies and microparticles were isolated by ultra-centrifugation. The treated EC, released microparticles and microparticle-free supernatant were analyzed for interleukin-1β (IL-1β) expression by western blot. In addition, we assessed the ability of microparticles derived from EC exposed to control IgG or anti-β₂GPI antibodies, or microparticle-free supernatant that had been adsorbed with immobilized β₂GPI to remove residual anti-β₂GPI antibodies, to activate EC. Activation was defined by increased expression of cell surface E-selectin and phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4).

Incubation of EC with β₂GPI and anti-β₂GPI antibodies stimulated the expression of pro-IL-1β. Interestingly, while EC contained exclusively pro-IL-1β, microparticles derived from these treated cells contained only the active, cleaved form of IL-1β. Moreover, extracellular IL-1β was found exclusively in microparticles, and was absent from microparticle-free conditioned medium Finally, IL-1β-containing microparticles derived from anti-β₂GPI antibody-treated EC, but not microparticles released from EC exposed to only control IgG activated additional EC. Conditioned medium from anti-β₂GPI antibody-treated EC that had been pre-adsorbed with immobilized β₂GPI to remove residual anti-β₂GPI antibodies also failed to stimulate EC activation.

EC exposed to β₂GPI and anti-β₂GPI antibodies stimulate the expression pro-IL-1β, which undergoes cleavage to an active form and is packaged in microparticles. These microparticles may directly induce activation of EC or synergize with anti-β₂GPI antibodies to activate EC. We hypothesize that delivery of IL-1β and perhaps other inflammatory cytokines by microparticles derived from anti-β₂GPI-exposed EC or monocytes may induce a systemic vascular inflammatory state in which anti-β₂GPI antibodies may more readily stimulate thrombotic events.

No relevant conflicts of interest to declare.