The release of microparticles by RAW 264.7 macrophage cells stimulated with TLR ligands.

MPs are small membrane-bound particles that originate from activated and dying cells and mediate intercellular communication. Once released from cells, MPs can serve as novel signaling elements in innate immunity, with levels elevated in immune-mediated diseases. This study tested the hypothesis that TLR stimulation can induce MP release by macrophages. In these experiments, using the RAW 264.7 murine macrophage cell line as a model, LPS, a TLR4 ligand, and poly(I:C), a TLR3 ligand, induced MP release effectively, as measured by flow cytometry; in contrast, a CpG oligonucleotide, which can stimulate TLR9, induced much lower levels of particle release. To determine the role of other mediators in this response, the effects of NO were tested. Thus, MP release from RAW 264.7 cells stimulated by LPS or poly(I:C) correlated with NO production, and treatment with the iNOS inhibitor 1400W decreased particle release and NO production. Furthermore, treatment of RAW 264.7 cells with NO donors induced MP production. As TLR ligands can induce apoptosis, the effect of caspase inhibition on MP release by stimulated cells was assessed. These experiments showed that the pan-caspase inhibitor, ZVAD, although decreasing NO production, increased MP release by stimulated cells. Together, these experiments demonstrate that TLR stimulation of macrophages can lead to MP release, and NO plays a key role in this response.