Importance of endothelial cell-leukocyte interactions in the sensing of TLR4 and NOD1 ligands by peripheral blood mononuclear cells in vitro

Grant Nicholson1, Laura Moreno1, Thomas Singer2, Harald Kropshofer2, Trevor Hansel1, Jane A. Mitchell1, C. Wheeler-Jones1. 1Imperial College, London, United Kingdom, 2Roche Pharmaceuticals, Basel, Switzerland.

Gram negative bacteria contain two important pathogen associated molecular patterns (PAMPs); lipopolysaccharide (LPS), which activates TLR4 receptors and peptidoglycan products, which activate NOD1 receptors. It is increasingly apparent that the innate immune system relies on amplification loops of cytokine networks and cell-cell interactions. Of particular relevance to innate immune sensing is the relationship between endothelial cells and leukocytes. In the current study we have investigated the consequence to the sensing of LPS or the NOD1 ligand, FK565, by endothelial cells and peripheral blood mononuclear cells (PBMCs) in isolation or in combination using co-culture systems. Cell activation was determined by measuring a range of nine inflammatory cytokines. Primary cultures of human umbilical vein endothelial cells (HUVECs) were plated in 96-well plates. PBMCs were prepared from blood of healthy donors and added to wells of 96-well plastic plates either alone or on top of confluent monolayers of HUVECs. Cells were stimulated either with a concentration range of LPS (data for 1μg/ml shown) or a maximal concentration of FK565 (100nM). After 20 hours at 37°C culture plates were centrifuged to obtain cell-free media samples and a range of cytokines measured (table 1).

Table 1: Cytokine release (pg/ml) from HUVEC, PBMC and HUVEC+PBMC cultures following 20 hours incubation with LPS (1μg/ml) or FK565 (10 nM). Data is the mean±S:E:M, n = 8 separate measures using PMBCs from 4 different donors. Responses to LPS and FK565 were compared to control samples using one-way ANOVA, followed by Dunnett’s post-test (* = P<0.05, ** = P<0.01).

LPS, but not FK565, increased release of all cytokines measured. Release of GM-CSF, IFN-γ and IL-6 was clearly enhanced when PBMCs were stimulated in the presence of HUVECs whilst IL-1β and TNF-α release was reduced in co-cultures compared to PBMCs alone. This data demonstrates the importance of PBMC:endothelial cell interactions in the sensing of PAMPs.

Supported by Roche.