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. 2009 Apr;126(4):475-84.
doi: 10.1111/j.1365-2567.2008.02922.x. Epub 2008 Sep 4.

Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells (V体育官网入口)

Rebecca J Dearman  1 , Marie Cumberbatch, Gavin Maxwell, David A Basketter, Ian Kimber
Affiliations

Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells

Rebecca J Dearman et al. Immunology. 2009 Apr.

Abstract

Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs VSports手机版. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses. .

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Figures

Figure 1
Figure 1
Influence of lipopolysaccharide (LPS) on dendritic cell (DC) maturation. DCs were harvested on day 6 or 8 of culture and incubated for 24 hr with LPS (serotype 055:B5; 100 ng/ml), or medium (med) alone. Membrane expression of the maturation markers major histocompatibility complex (MHC) class II, CD80, CD86, CD40 and CD54 was assessed by flow cytometric analysis of 104 cells. (a) Representative histograms showing the pattern of expression of each of the membrane markers (—) versus isotype control antibody (- - -) for cells cultured on day 6 or 8 with LPS or medium alone. A representative experiment is shown for days 6 and 8. (b) Mean fluorescence intensity (MFI) recorded for each membrane marker following culture of day 6 (▪) or day 8 (□) DCs in the presence of LPS or medium alone. The mean ± standard error of four independent experiments is shown. The statistical significance of differences between medium alone and LPS-treated groups was P < 0.05, with the exception of MHC class II for day 8 DCs.
Figure 2
Figure 2
Lipopolysaccharide (LPS) induces cytokine secretion by dendritic cells (DCs). DCs were harvested on day 6 (▪) or day 8 (□) of culture and incubated for 24 hr with LPS (serotype 055:B5; 100 ng/ml), or medium alone (med). Supernatants were harvested and cytokine content measured by cytokine protein bead array. Results are displayed as mean cytokine content (± standard error) derived from five independent experiments for day 6 DCs and seven independent experiments for day 8 DCs. The statistical significance of differences between medium alone and LPS-treated groups was P < 0.05 for all cytokines.
Figure 3
Figure 3
Toll-like receptor (TLR) ligands induce maturation of dendritic cells (DCs). DCs were harvested on day 6 of culture and incubated for 24 hr with peptidoglycan (PG; 100 μg/ml), zymosan (Zy; 100 μg/ml), Pam3Cys-Ser-(Lys)4 (PAM; 100 ng/ml), macrophage-activating lipopeptide-2 (MALP-2; 100 ng/ml), lipopolysaccharide (LPS) R515 (100 ng/ml), flagellin (500 ng/ml) and CpG (5 μg/ml). Expression of the membrane markers CD40 and CD86 was determined by flow cytometric analysis of 104 cells. Results are displayed as mean fluorescence intensity (MFI) recorded for each marker. Results from two independent experiments (▪, □) are shown.
Figure 4
Figure 4
Influence of Toll-like receptor (TLR) ligands on cytokine secretion by DCs: a dose–response analysis. DCs were harvested on day 6 of culture and were incubated for 24 hr with increasing concentrations of peptidoglycan (PG; • 1, 10 and 100 μg/ml), zymosan (Zy; ○; 1, 10 and 100 μg/ml), Pam3Cys-Ser-(Lys)4 (PAM; ▪; 1, 10 and 100 ng/ml), macrophage-activating lipopeptide-2 (MALP-2; □; 1, 10 and 100 ng/ml), lipopolysaccharide (LPS) R515 (▴; 1, 10 and 100 ng/ml), flagellin (♦; 100, 200 and 500 ng/ml) or CpG (Δ; 1, 2, 5 and 10 μg/ml) or in the absence of TLR ligand (⋊). Supernatants were harvested and cytokine content determined by cytokine protein bead array. Representative results from one independent experiment are displayed. IL, interleukin; TNF, tumour necrosis factor.
Figure 5
Figure 5
Different Toll-like receptor (TLR) ligands induce distinct patterns of cytokine secretion by DCs. DCs were harvested on day 6 of culture and incubated for 24 hr with peptidoglycan (PG; 100 μg/ml), zymosan (Zy; 100 μg/ml), Pam3Cys-Ser-(Lys)4 (PAM; 100 ng/ml), macrophage-activating lipopeptide-2 (MALP-2; 100 ng/ml), lipopolysaccharide (LPS) R515 (100 ng/ml), flagellin (500 ng/ml), CpG (5 μg/ml), or medium (med) alone. Supernatants were harvested and cytokine content determined by cytokine protein bead array. Results are displayed as mean cytokine content (± standard error) derived from three independent experiments for PAM, MALP-2, LPS R515, flagellin and CpG, and mean cytokine content (± range) of two separate experiments for PG and Zy. The statistical significance of differences between medium alone and TLR ligand-treated groups was P < 0·05 for PAM, MALP-2, LPS R515 and CpG for all cytokines, and for flagellin for IL-1β and IL-12 p40 only.
Figure 6
Figure 6
Toll-like receptor (TLR) ligands provoke dose-dependent increases in chemokine secretion by dendritic cells (DCs). DCs were harvested on day 6 of culture and were incubated for 24 hr with increasing concentrations of peptidoglycan (PG; •; 1, 10 and 100 μg/ml), zymosan (Zy; ○; 1, 10 and 100 μg/ml), Pam3Cys-Ser-(Lys)4 (PAM; ▪; 1, 10 and 100 ng/ml), macrophage-activating lipopeptide-2 (MALP-2; □; 1, 10 and 100 ng/ml), lipopolysaccharide (LPS) R515 (▴; 1, 10 and 100 ng/ml), flagellin (♦; 100, 200 and 500 ng/ml) or CpG (Δ; 1, 2, 5 and 10 μg/ml) or in the absence of TLR ligand (⋄). Supernatants were harvested and chemokine content determined by chemokine protein bead array. Representative results from one independent experiment are displayed. MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; KC, keratinocyte-derived chemokine; MIG, monokine induced by interferon-γ; IP, interferon-γ inducible protein.
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