Introduction

Toll-like receptor 4 (TLR4) and the associated MD-2 protein play a role in the response of myeloid cells to LPS. Antibodies to TLR4 have been reported to inhibit the LPS-induced cytokine production by human PBMCs and mouse peritoneal macrophages. We have modified the blocking experiments published in the references below with monoclonal antibodies HTA125 (anti-human TLR4) and MTS510 (anti-mouse TLR4/MD2). This protocol provides a general guideline; investigators should optimize experimental conditions including cell type, density, antibody concentration, ligands and kinetics.

Important: We have found the strain of LPS listed below to provide the best stimulation for cytokine production in the assays used in our laboratory. If there are additional sources of stimulation including other bacterial products in the assay media or reagents used, or high levels of cytokine secretion by cells in medium alone, it will be difficult to observe significant level of inhibition by anti-TLR4 antibodies.

Anti-TLR4 Neutralization of LPS-Induced Cytokine Production by Human PBMC

Materials

• Human PBMC suspension
• Culture Medium (RPMI supplemented with 10% FBS)
• 24-well flat-bottom culture plate (Costar Cat. No. 3526)
• Functional Grade (FG) anti-human TLR4, Clone HTA125 (Cat. No. 16-9917)
• Functional Grade (FG) mouse IgG2a isotype control (Cat. No. 16-4724)
• Lipopolysaccharide (LPS) (Sigma, Cat. No. L-8274)
• eBioscience Human IL-6 Ready-SET-Go! ELISA set (Cat. No. 88-7066), or eBioscience Human TNF-α Ready-SET-Go! ELISA set (Cat. No. 88-7346)

Experiment Duration

• 1 hour assay preparation
• 16-24 hour incubation
• Time required for ELISA Ready-SET-Go!

Method

1. Prepare human PBMCs, dispense 1ml at 2x10⁶/ml into each well of a 24-well culture plate.
2. Pre-incubate cells with 20µg/ml of HTA125 or isotype control antibody.
3. Incubate for 0.5-1 hour at 37°C.
4. Add LPS at a concentration of 0.1ng/ml.
5. Incubate cells for 16-24 hours at 37°C, 5% CO₂ in a humidified incubator.
6. Harvest 1ml of media from each well, spin at 500xg, collect supernatant and discard the cell pellet.

Anti-TLR4/MD2 Neutralization of Lipid A-Induced Cytokine Production by Mouse Peritoneal Cells

Materials

• Thioglycolate elicited mouse peritoneal cell suspension
• Culture Medium (RPMI supplemented with 10% FBS)
• 24-well flat-bottom culture plate (Costar Cat. No. 3526)
• Functional Grade (FG) anti-mouse TLR4/MD2, Clone MTS510 (Cat. No. 16-9924)
• Functional Grade (FG) rat IgG2a isotype control (Cat. No. 16-4321)
• Lipid A (Sigma, Cat. No. L-0774)
• eBioscience Mouse IL-6 Ready-SET-Go ELISA set (Cat. No. 88-7064), or eBioscience Mouse TNF-α Ready-SET-Go ELISA set (Cat. No. 88-7324)

Experiment Duration

• 1 hour assay preparation
• 16-24 hour incubation
• Time required for ELISA Ready-SET-Go!

Method

1. Prepare peritoneal cells and dispense 1ml at 2x10⁶/ml into each well of a 24-well culture plate.
2. Pre-incubate cells with 20µg/ml of MTS510 or isotype control antibody.
3. Incubate for 0.5-1 hour at 37°C.
4. Add Lipid A at a concentration of 5ng/ml.
5. Incubate cells for 16-24 hours at 37°C, 5% CO₂ in a humidified incubator.
6. Harvest 1ml of media from each well, spin at 500xg, collect supernatant and discard the cell pellet.

References

1. Akashi S, et al. 2000. Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2. Biochem Biophys Res Commun. 268(1): 172-7.
2. Akashi S, et al. 2000. Cell surface expression and lipopolysaccharide signaling via the Toll-like receptor 4-MD2 complex on mouse peritoneal macrophages. J. Immunol. 164: 3471-5.
3. Tabeta K, et al. 2000. Toll-like receptors confer responsiveness to lipopolysaccharide from Porphyromonas gingivalis in human gingival fibroblasts. Infect Immun. 68(6): 3731-5.