Toll-like receptor 9-dependent and -independent dendritic cell activation by chromatin-immunoglobulin G complexes

Abstract

Dendritic cell (DC) activation by nucleic acid-containing immunoglobulin (Ig)G complexes has been implicated in systemic lupus erythematosus (SLE) pathogenesis. However, the mechanisms responsible for activation and subsequent disease induction are not completely understood. Here we show that murine DCs are much more effectively activated by immune complexes that contain IgG bound to chromatin than by immune complexes that contain foreign protein. Activation by these chromatin immune complexes occurs by two distinct pathways. One pathway involves dual engagement of the Fc receptor FcgammaRIII and Toll-like receptor (TLR)9, whereas the other is TLR9 independent. Furthermore, there is a characteristic cytokine profile elicited by the chromatin immune complexes that distinguishes this response from that of conventional TLR ligands, notably the induction of BAFF and the lack of induction of interleukin 12. The data establish a critical role for self-antigen in DC activation and explain how the innate immune system might drive the adaptive immune response in SLE.

Figure 1.

Chromatin IC and protein IC bind DCs comparably. (a) Protein IC (anti-TNP IgG2a mAb plus TNP-BSA) and chromatin IC (antinucleosome IgG2a mAb plus spent culture fluid) were tested for immune complex formation in a C1q binding assay and compared with monomeric or heat-aggregated (HA) mouse IgG. (b) FACS^® analysis of bone marrow–derived DCs from wild-type C57BL/6 mice purified with anti-CD11c magnetic beads. (c) The ability of the monomeric anti-TNP mAb (α-TNP mAb) and the immune complexes described above to bind to bone marrow–derived DCs from wild-type C57BL/6 mice was compared by measuring the amount of IgG2a bound to the cell surface using flow cytometry.

Figure 2.

Chromatin IC, but not protein IC, induce TNF-α production. Bone marrow–derived DCs from wild-type C57BL/6 mice were preincubated for 30 min with or without (a) 12 μg/ml inhibitory CpG sODN 2088, (b) sODN 2138, sODN 1982, sODN 2088 (all at 1 μg/ml), and (c) 20 μg/ml chloroquine before the addition of 50 μg/ml protein IC, 50 μg/ml chromatin IC (PL2-3, IgG2a; PL2-8, IgG2b), 6 μg/ml of the stimulatory CpG sODN 1826, 10 μg/ml LPS, and 100 μg/ml poly(I:C). TNF-α and IL-12 p70 concentrations in supernatants collected after 48 h were determined by ELISA. The data are representative of nine (a), three (b), and three experiments (c).

Figure 3.

Chromatin IC–induced TNF-α production involves MyD88 and TLR9. Bone marrow–derived DCs from wild-type C57BL/6 and MyD88-deficient mice (left), or wild-type BALB/c and TLR9-deficient mice (right), were cultured with protein IC, chromatin IC (PL2-3, IgG2a; PL2-8, IgG2b), the stimulatory CpG sODN 1826, and LPS. TNF-α concentrations in supernatants collected after 48 h were determined by ELISA. Data represent mean + SEM of three (left) and five experiments (right).

Figure 4.

FcγRIII is required for chromatin IC–induced TNF-α production. Bone marrow–derived DCs from wild-type C57BL/6, Fc receptor common γ chain–deficient (FcRγ chain −/−), FcγRIII-deficient (FcγRIII−/−), and FcγRII-deficient (FcγRII−/−) mice were cultured with protein IC, chromatin IC (PL2-3, IgG2a; PL2-8, IgG2b), the stimulatory CpG sODN 1826, and LPS. TNF-α and IL-12 p70 concentrations in supernatants collected after 48 h were determined by ELISA. The data are representative of three experiments.

Figure 5.

BAFF induction by chromatin IC is TLR9 independent. Bone marrow–derived DCs from wild-type BALB/c and TLR9-deficient mice were cultured with protein IC, chromatin IC (PL2-3, IgG2a; PL2-8, IgG2b), the stimulatory CpG sODN 1826, and LPS. BAFF concentration in supernatants collected after 48 h was determined by ELISA. The data are representative of three experiments.

Figure 6.

Nucleosome/antinucleosome complexes activate DCs. (a) Bone marrow–derived DCs from wild-type BALB/c and TLR9-deficient mice were cultured with the stimulatory CpG sODN 1826, LPS, R848, nucleosome fraction 4 (nucleosome 4; mainly mononucleosomes), nucleosome fraction 7 (nucleosome 7; mainly di-nucleosomes and tri-nucleosomes), the antinucleosome IgG2a mAb PL2-3, PL2-3 and nucleosome 4, PL2-3 and nucleosome 7, protein IC (anti-TNP IgG2a mAb plus TNP-BSA), and protein IC and nucleosome 4. TNF-α concentrations in supernatants collected after 24 h were determined by ELISA. The data are representative of three experiments. (b) Day 6 bone marrow–derived DC cultures (not CD11c purified) from wild-type BALB/c and TLR9-deficient mice were cultured with stimuli as shown. After 24 h of incubation, DCs were double stained with anti–CD11c-PE and anti–CD86-FITC. CD86 expression of the CD11c⁺ population gate is shown with stimulus-induced staining intensity (black) compared with staining intensity of the nonstimulated cultures (gray). The data are representative of three experiments.