TNF-alpha release from primary human PBMC
Peripheral blood mononuclear cells (PBMC) play an important role in the immune system and the typical population consists of T cells (CD4 and CD8 positive, approximately 75%), B cells and NK cells (25% combined).This assay utilizes the endotoxin lipopolysaccharide (LPS) as a strong inducer of an inflammatory response. LPS activates the Toll-like Receptor 4 (TLR4) resulting in the release of the pro-inflammatory cytokine TNF-alpha. The test compound’s ability to inhibit TNF-alpha release is measured. Adverse effects (undesired compound effects) can also result in a reduction of TNF-alpha, for example: a cytotoxic compound will decrease TNF-alpha levels by simply reducing cell viability and cell number, but via a non-desired mechanism. In order to distinguish a specific inhibition from a cytotoxic effect a secondary readout, cell viability, is provided.
Assay protocol outline
This ‘classic’ inflammatory assay utilizes primary human PBMC activated by 10 ng/ml LPS in 384 well format (5000vcells/well). After incubation for 24 hours, the level of released TNF-alpha is measured. Compounds are tested for the ability to inhibit the LPS-induced TNF-alpha release. Cellular viability is used to evaluate possible cytotoxicity.
The figure illustrates the potent effect of the steroid Dexamethasone (blue curve) inhibiting the detectable levels of TNF-alpha by 90% efficacy (Emax). Calculated relative EC50 (Effective Concentration at 50% effect) for Dexamethasone is approximately 25 nM. The green curve shows the effect of Calcipotriol on the release of TNF-alpha to be approximately 0.2 nM with Emax at around 40%. A toxic compound will reduce cell viability while a specific TNF-alpha release inhibitor will not. In general it is preferable to have a potent and efficacious compound which reduces the TNF-alpha levels without affecting cell viability. The EC50 and Emax values are calculated using a 4-parameter sigmoidal curve-fitting algorithm. Y-axis shows effect of TNF-alpha inhibition in percent (%). Neither Dexamethasone nor Calcipotriol have significant effects on the cell viability.