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 e.g. 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. E.g. 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 and reports the 'functionality ' of the cells.
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 levelof 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 detected 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 a max effect (Emax) at around 40%. A toxic compound will reduce cell viability while a specific TNF-alpha release inhibitor will not. In general it is preferred to have a potent and efficacious compound which reduced the TNF-alpha levels without effect on the 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 (%). Nor Dexamethasone or Calcipotriol have any significant effect on the cell viability.