Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins

Abstract

The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3alpha (MIP-3alpha), and the MyD88-independent molecules beta interferon (IFN-beta), nitric oxide, and IFN-gamma-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-alpha, IL-1beta, and MIP-3alpha, but significantly less IFN-beta, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-beta, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-alpha and MIP-3alpha in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-alpha release but did not influence nitric oxide release. IFN-beta polyclonal antibody and IFN-alpha/beta receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.

FIG. 1.

TNF-α and MIP-3α (MyD88-dependent) induction by endotoxin. Neisseria meningitidis LOS and E. coli 55:B5 and Vibrio cholerae LPSs induced more TNF-α and MIP-3α compared to Serratia marcescens, Salmonella enterica serovar Minnesota, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa LPSs. *, P < 0.001. TNF-α (solid bars) and MIP-3α (dashed bars) induction in 10⁶ THP-1 cells stimulated with highly purified LOS or LPS (0.56 pmol lipid A/ml) for 18 h is shown. Error bars represent standard deviation from the average of four readings. The figure is representative of six independent experiments.

FIG. 2.

Nitric oxide and IP-10 (MyD88-independent) induction by endotoxin. N. meningitidis LOS and Salmonella LPS induced significantly more nitric oxide compared to other endotoxins (P < 0.001). A: RAW 264.7 cells (10⁶ cells/ml) stimulated directly (solid bars) with highly purified LPS (0.56 pmol lipid A/ml) or indirectly (dashed bars) with 50 μl/well of THP-1 supernatants from cells stimulated with 0.56 pmol lipid A/ml LPS and incubated overnight. Nitric oxide release was determined by measuring nitrite accumulated in supernatants with the Greiss method. Error bars represent standard deviation from the average of four readings. Data are representative of eight independent experiments. B: IP-10 induction by endotoxins. THP-1 (10⁶ cells/ml) were stimulated with 0.56 pmol lipid A/ml of highly purified endotoxins and incubated for 18 h. IP-10 release was quantified in harvested supernatants using an ELISA. Error bars represent standard deviation from the average of four readings. Data are representative of four independent experiments. All P values were *, < 0.0001 compared to N. meningitidis LOS or **, < 0.005 compared to S. enterica serovar Typhimurium or S. enterica serovar Minnesota LPS.

FIG. 3.

Inhibition of MyD88-dependent signaling pathway with DNMyD88. A: TNF-α induction from THP-1 cells alone (solid bars) or cells transfected with DNMyD88 (dashed bars) stimulated with 5.6 pmol lipid A/ml of endotoxin and incubated for 18 h (P < 0.001). B: Indirect nitric oxide induction in RAW 264.7 cells stimulated with 50 μl of supernatants harvested from cells in panel A. Error bars represents the standard deviation from the average of 4 readings. This figure is representative of three independent experiments. Similar results were obtained when the experiments were performed at higher (112 pmol/ml) and lower (0.56 pmol/ml) endotoxin doses.

FIG. 4.

Dose-dependent induction of TNF-α and nitric oxide release by endotoxin. A: TNF-α induction from THP-1 cells stimulated with serial fold dilutions of N. meningitidis LOS and S. enterica serovar Typhimurium, E. coli 55:B5 and V. cholerae LPSs (10-0.0046 pmol/ml) and incubated for 18 h. B: Direct nitric oxide induction from RAW 264.7 cells stimulated with serial fold dilutions of endotoxins (10-0.0046 pmol lipid A/ml) and incubated for 18 h. C: Indirect nitric oxide induction in RAW 264.7 cells stimulated with serial fold dilutions of supernatants (50 μl to 0.046 μl/well) harvested from THP-1 cells previously stimulated with 0.56 pmol lipid A/ml LPS for 2 h. Error bars represent the ± standard deviation from the average of 4 readings. *, P values indicate significant differences compared to N. meningitidis LOS. This figure is representative of six independent experiments. N. meningitidis LOS, diamonds; S. enterica serovar Typhimurium LPS, squares; E. coli 55:B5 LPS, triangles; V. cholerae LPS, crossed squares.

FIG. 5.

Time course induction of the MyD88-dependent and -independent signaling pathways by endotoxin. A: Induction of TNF-α from THP-1 (10⁶ cells/ml) stimulated with 0.56 pmol lipid A/ml of endotoxin overnight. B: Indirect nitric oxide induction from RAW 264.7 cells stimulated with 50 μl of the same THP-1 supernatants harvested in panel A. N. meningitidis LOS, diamonds; S. enterica serovar Typhimurium LPS, squares; E. coli 55:B5 LPS, triangles; V. cholerae LPS, crossed squares. Error bars represent the standard deviation from the average of four readings. This figure is representative of six independent experiments.

FIG. 6.

Induction of nitric oxide by endotoxin was IFN-β mediated. A: Nitric oxide induction in RAW 264.7 cells stimulated with two fold dilutions (3-0.19 ng/ml) of exogenous IFN-β. B: Nitric oxide induction in RAW 264.7 cells stimulated with 50 μl supernatants harvested from THP-1 cells (10⁶) blocked with 1, 5, or 10 μg of anti-IFN-α/β receptor 1 polyclonal antibody and stimulated with 10 pmol/ml of N. meningitidis lipid A. Controls were supernatants (50 μl) from unstimulated THP-1 cells, unstimulated RAW 264.7 cells, and lipid A without anti-IFN-α/β receptor 1 antibody. C: Direct nitric oxide induction in RAW 264.7 cells stimulated with decreasing doses of wild-type N. meningitidis LOS in the presence or absence of murine anti-IFN-β polyclonal antibody (2 μg/well). D: Indirect nitric oxide induction in RAW 264.7 cells induced with 50 μl of THP-1 supernatants stimulated with 10 pmol/ml of N. meningitidis lipid A. Anti-IFN-α (2 μg/well) or anti-IFN-β (2 μg/well) or both antibodies together were added directly into RAW 264.7 cells. Error bars represent the standard deviation from the average of four readings. This experiment figure is representative of three independent experiments.