Differential roles of Toll-like receptors in the elicitation of proinflammatory responses by macrophages

Abstract

BACKGROUND—Mammalian Toll-like receptor (TLR) proteins are pattern recognition receptors for a diverse array of bacterial and viral products. Gram negative bacterial lipopolysaccharide (LPS) activates cells through TLR4, whereas the mycobacterial cell wall glycolipids, lipoarabinomannan (LAM) and mannosylated phosphatidylinositol (PIM), activate cells through TLR2. Furthermore, short term culture filtrates of M tuberculosis bacilli contain a TLR2 agonist activity, termed soluble tuberculosis factor (STF), that appears to be PIM. It was recently shown that stimulation of RAW264.7 murine macrophages by LPS, LAM, STF, and PIM rapidly activated NF-κB, AP1, and MAP kinases.
RESULTS—This study shows that signalling by TLR2 and TLR4 also activates the protein kinase Akt, a downstream target of phosphatidylinositol-3'-kinase (PI-3-K). This finding suggests that activation of PI-3-K represents an additional signalling pathway induced by engagement of TLR2 and TLR4. Subsequently, the functional responses induced by the different TLR agonists were compared. LPS, the mycobacterial glycolipids, and the OspC lipoprotein (a TLR2 agonist) all induced macrophages to secrete tumour necrosis factor α (TNFα), whereas only LPS could induce nitric oxide (NO) secretion. Human alveolar macrophages also exhibited a distinct pattern of cellular response after stimulation with TLR2 and TLR4 agonists. Specifically, LPS induced TNFα, MIP-1β, and RANTES production in these cells, whereas the TLR2 agonists induced only MIP-1β production.
CONCLUSION—Together, these data show that different TLR proteins mediate the activation of distinct cellular responses, despite their shared ability to activate NF-κB, AP1, MAP kinases, and PI-3-K.



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Figure 1  .

TLR2 is required for macrophage activation by mycobacterial glycolipids. Adherent peritoneal macrophages elicited by thioglycollate were collected from TLR2−/− and C57BL/6 (WT) mice and stimulated for 24 hours with LPS (100 ng/ml), STF (20 µl/ml), LAM (1 µg/ml), and PIM (1 µg/ml). Supernatants were collected and concentrations of secreted TNFα were determined by ELISA. Assays were performed in triplicate and repeated on three separate occasions. A single representative experiment is shown and data are expressed as mean values (SEM). LPS = lipopolysaccharide; STF = soluble tuberculosis factor; LAM = lipoarabinomannan; PIM = phosphatidylinositol dimannoside.

Figure 2  .

TLR2 agonists fail to induce nitric oxide production. LPS and the mycobacterial glycolipids were analysed for their abilities to induce TNFα and nitric oxide (NO) production in the murine macrophage-like cell line RAW264.7. NO levels were indirectly determined by measuring the levels of the stable NO catabolite nitrite in the culture supernatants of stimulated macrophages. Cells were stimulated for 24 hours with LPS (100 ng/ml), STF (20 µl/ml), LAM (5 µg/ml), lipoprotein OspC (100 ng/ml), and PIM (5 µg/ml). Supernatants were collected and analysed for the presence of nitrite using the Greiss assay, and for TNFα by ELISA. Assays were performed in triplicate and repeated on three separate occasions. A single representative experiment is shown and data are expressed as mean values (SEM). LPS = lipopolysaccharide; STF = soluble tuberculosis factor; LAM = lipoarabinomannan; PIM = phosphatidylinositol dimannoside.

Figure 3  .

Both TLR2 and TLR4 agonists induce Akt activation in a PI-3-K dependent manner. RAW264.7 cells were pretreated for one hour with either the PI-3-K inhibitor LY294002 (25 µM) or vehicle (DMSO, 6.5 µl/ml), and then stimulated with either LPS (100 ng/ml) or PIM (4 µg/ml), for 30 minutes. After stimulation, whole cell lysates were prepared, fractionated by SDS-PAGE (50 µg lysate/lane), and transferred to nitrocellulose membranes. The membranes were then probed with antibodies against the phosphorylated forms of Akt and the p38 MAP kinase. Duplicate membranes were also probed with antibodies specific for the non-phosphorylated forms of Akt and p38. Bound primary antibodies were detected using secondary antibodies conjugated to HRP. LPS = lipopolysaccharide; PIM = phosphatidylinositol dimannoside.

Figure 4  .

Interferon gamma (IFNγ) confers on TLR2 agonists the ability to induce NO production. RAW264.7 macrophages were pretreated for one hour with 10 ng/ml of murine IFNγ and then stimulated for 24 hours with LPS (100 ng/ml), STF (20 µl/ml), or LAM (5 µg/ml). Supernatants were collected and analysed for NO using the Greiss assay. Assays were performed in triplicate and repeated on three separate occasions. A single representative experiment is shown and data are expressed as mean values (SEM). LPS = lipopolysaccharide; STF = soluble tuberculosis factor; LAM = lipoarabinomannan.