Whole Blood Assay LPS

Contact:
Louise Berg
Louise.Berg@ki.se
Rationale

In the chronic inflammatory diseases, tissues such as skin, lung, joints are invaded by inflammatory leukocytes. Tissue inflammation depends on activation of endothelial cells, leukocyte rolling , adherence and transmigration. The transmigration of leukocytes into the tissue depends on chemoattractants, of which IL8 is a potent chemoattractant and activator of neutrophils. It can be produced by monocytes and by endothelial cells, among other celltypes.

Aim

To investigate whether chemical inhibitory probes can inhibit IL8 secretion induced by stimulation of whole blood with lipopolysaccharide (LPS).

Conclusions

We found inhibitory effects using MEK1/2 inhibitors, as well as one ERK inhibitor, on LPS induced IL8 secretion. This agrees with already published reports. None of the other 56 probes showed consistent inhibitory effects in this assay.

Experimental Protocol

General protocol: Whole heparinized blood is cultured for 24 hours in presence of LPS 10ug/ml. The effect on IL8 secretion by addition of chemical probes is investigated by ELISA.

Cell culture condition: 225ul heparinised blood, in triplicates, is dispensed into a 96 well plate and pre-incubated with 25ul probes (1μM or 0,1μM) 30 min before addition of LPS 10ug/ml in 10ul. Plate is incubated 24h whereafter it is centrifuged and 100ul supernatant removed and stored at -20C until analysis.

Readout: Cell culture supernatants are removed after 24 hours and analysed for IL8 using ELISA (MabTech cat#3560-1H). Controls include unstimulated condition (without LPS), LPS-stimulated condition with vehicle control only (DMSO), stimulated condition with IRAK 1/4 inhibitor I.

IL8 release upon 24 hours stimulation LPS was investigated in 13 blood donors including 8 patients with systemic lupus erythemtosus (SLE), 3 with dermatomyositis (DM) and 2 healthy controls (HC). LPS induced variable levels of IL8. DMSO had little effect on the IL8 released, except for HC28. In both healthy donors (HC28, HC27) the variation between biological triplicates were great, making interpretation of data from these donors difficult.

Fig 1

IL8 released after 24h stimulation of whole blood with LPS, in absence and presence of DMSO (vehicle control). Data are depicted from 13 individual blood donors, as average+SD of biological triplicates.

 

A set of 58 probes were tested in 13 blood donors including 8 patients with systemic lupus erythemtosus (SLE), 3 with dermatoymyositis (DM) and 2 healthy donors. Not all probes were tested in every blood donor (white boxes=no data). 5-13 donors were tested for each probe. The effect on whole blood IL8 secretion after 24h cell culture in presence of LPS is expressed normalized to the vehicle control (0,01% or 0,001% DMSO). Probes are added at 1uM or 0,1uM, as indicated. The MEK1/2 inhibitor Trametinib and the ERK inhibitor SCH772984 were found to have inhibitory effects (p<0,001).

Log2 transformed
White box: no data available

Fig 2

To validate the effects observed using the MEK1/2 inhibitor Trametinib, we tested two other MEK inhibitors. Due to the lack of alternative high quality ERK inhibitors, the effect seen using the ERK 1/2 inhibitor SCH772984 was not further studied. The MEK 1,2 inhibitor PD0325901 and the MEK 1 inhibitor Selumetinib also had inhibitory effects (n=3, average+SD of biological triplicates). Thus, we confirm that MEK is involved in LPS mediated IL8 secretion.

 

Fig 3