Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens

Abstract

Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens.

Figure 2

Quantitation of TLR mRNA by real-time quantitative RT-PCR in freshly isolated monocytes (Mono), CD11c⁺ imDCs, and plasmacytoid pre-DCs (pDC), and in immature and mature DCs induced from them. Monocytes were cultured with 50 ng/ml GM-CSF and 200 U/ml IL-4 for 5 d to obtain imDC1 and were further stimulated with CD40L-transfected L cells for 24 h to obtain mDC1. CD11c⁺ imDCs were stimulated with CD40L-transfected L cells for 24 h to obtain mature CD11c⁺ DCs. Plasmacytoid pre-DCs were cultured with 10 ng/ml IL-3 for 5 d to obtain imDC2 and were further stimulated with CD40L-transfected L cells for 24 h to obtain mature DC2. The amounts of mRNA were quantitated by real-time quantitative RT-PCR, and were shown by arbitrary unit relative to the amount of ubiquitin mRNA. The data shown are representative of three experiments.

Figure 1

Detection of TLR mRNA by RT-PCR in freshly isolated monocytes, CD11c⁺ imDCs, and plasmacytoid pre-DCs. The three cell populations were isolated by cell sorting to the purity of >99%. cDNAs were amplified for 35 cycles and were separated on a 3% agarose gel containing ethidium bromide. The data shown are representative of three experiments.

Figure 3

TNF-α, IL-6, IFN-α, and IL-12 production by monocytes (Mono), CD11c⁺ imDCs, and plasmacytoid pre-DCs (pDC) stimulated with microbial molecules that trigger different TLR signaling. The three cell populations were cultured with 10 μg/ml PGN, 10 μg/ml LTA, 10 μg/ml LPS, 50 μg/ml poly I:C, or with 5 μM CpG-ODN AAC-30 for 24 h, and the concentrations of cytokines in the supernatants were measured by ELISA. The data shown are representative of four or five experiments.