Primary blood neutrophils express a functional cell surface Toll-like receptor 9.

Polymorphonuclear leukocytes (PMNs) represent one of the first lines of defense against pathogens. TLR9 is normally expressed in endosomes/lysosomes where it is activated by pathogen-derived DNA. Here we show that freshly isolated human and mouse primary PMNs express TLR9 at the cell surface ex vivo. Moreover, surface TLR9 expression is upregulated upon activation of PMNs with different stimuli and not only TLR9 agonists. Importantly, surface TLR9 is processed, active, and functional. TLR9 ligands, oligo-nucleotides containing unmethylated CpG motifs, indeed bind to surface TLR9 and binding was strongly observed at the cell surface of human cells expressing surface TLR9 and at the surface of WT but not TLR9-deficient mouse PMNs. Finally, CpG oligonucleotides cross-linked onto a solid phase and having no access to intracellular TLR9 are able to trigger cell surface TLR9 and induce neutrophil activation, even when endosomal acidification is inhibited. This is the first demonstration of a functional TLR9 expressed at the cell surface of human primary cells. This pathway may be triggered when pathogen-derived TLR9 ligands cannot reach the endosome, offering a rescue mechanism for neutrophil activation.

TLR2/TLR4-independent neutrophil activation and recruitment upon endocytosis of nucleosomes reveals a new pathway of innate immunity in systemic lupus erythematosus.

The nucleosome is a major autoantigen in systemic lupus erythematosus (SLE); it can be detected as a circulating complex in the serum, and nucleosomes have been suggested to play a key role in disease development. In the present study, we show for the first time that physiological concentrations of purified nucleosomes trigger innate immunity. The nucleosomes are endocytosed and induce the direct activation of human neutrophils (polymorphonuclear leukocytes (PMN)) as revealed by CD11b/CD66b up-regulation, IL-8 secretion, and increased phagocytic activity. IL-8 is a neutrophil chemoattractant detected in high concentrations in the sera of patients, and IL-8 secretion might thus result in enhanced inflammation, as observed in lupus patients, via an amplification loop. Nucleosomes act as free complexes requiring no immune complex formation and independently of the presence of unmethylated CpG DNA motifs. Both normal and lupus neutrophils are sensitive to nucleosome-induced activation, and activation is not due to endotoxin or high-mobility group box 1 contamination. In mice, i.p. injection of purified nucleosomes induces neutrophil activation and recruitment in a TLR2/TLR4-independent manner. Importantly, neutrophils have been suggested to link innate and adaptive immunity. Thus, nucleosomes trigger a previously unknown pathway of innate immunity, which may partially explain why peripheral tolerance is broken in SLE patients.