Extracellular ATP Acting Via P2 Receptors Enhances Pro-inflammatory Cytokine Secretion From Human Lung Parenchyma: An Important Role In Allergic Asthma?

Kylie Such1, Ernesto Oviedo-Orta1, David Hall2, Susanna Hourani1. 1University of Surrey, Guildford, UK, 2GlaxoSmithKline, Stevenage, UK

ATP acting via P2 receptors is widely recognised as a ‘danger signal’ in the immune system. ATP released from damaged cells during inflammation causes the maturation of dendritic cells which activate an antigen specific T-cell response to the invading antigen (la Sala et al. 2003). This could play a role in allergic asthma, where the generation of an antigen specific T-cell response is the first step to allergic sensitisation. Patients with asthma have increased inflammatory responses to ATP (Basoglu et al. 2005), and the presence of P2 receptors on immune cells could indicate a role for ATP in inflammatory diseases. The aim of this study was to investigate the effects of various nucleotides in combination with bacterial lipopolysaccharide (LPS) on the immune response in human lung parenchyma. Post-mortem human lungs, which were not suitable for transplantation, were obtained from the National Disease Research Interchange (Philadelphia) under the US Uniform Anatomical Gifts Act. Consent for research was obtained from the next of kin. Lung tissue was chopped and sieved to <1mm fragments, incubated in RPMI (plus 10% FCS, penicillin 100u/ml, streptomycin 100µg/ml and glutamate 2mM) at 37°C plus 5% CO2 and stimulated for 24hours with LPS in the presence or absence of nucleotides at 1000, 330, 110, 37, 12 and 4µM. Supernatants were removed and analysed for cytokine levels via MSD assay. Data are expressed relative to the LPS value to account for inter-individual variation in cytokine levels. Data are reported as %LPS (mean ± s.e.m of 5 independent experiments) and were compared for statistical analysis by 2 way ANOVA with post hoc Bonferroni test.

ATPγS caused a concentration-dependant increase in the pro-inflammatory cytokine IL-1β eliciting a 9800±4000% increase at 1000µM compared to LPS alone (p<0.001; baseline cytokine level 396±129pg/ml, maximum stimulated level 11483±3353pg/ml). ATPγS also induced a concentration-dependant increase in IL-17 (400±60% at 1000µM, p<0.001; baseline 39±23pg/ml, maximum 99±18pg/ml) and IFNγ (560±200% at 1000µM, p<0.01; baseline 450±417pg/ml, maximum 244±187pg/ml). UTP also caused a concentration-dependant increase in IFNγ release (490±210% at 1000µM, p<0.05; baseline 201±170pg/ml, maximum 190±174pg/ml). TNFα release was decreased by 1000µM ATPγS (by 50±8%, p<0.05; baseline 633±392pg/ml, minimum cytokine level 414±339pg/ml), and αβmethyleneATP (by 34±3%, p<0.05; baseline 1506±1335pg/ml, minimum level 454±392pg/ml), and increased by 1000µM AMP (by 86±30%, p<0.005; baseline 1933±618pg/ml, maximum 2304±578pg/ml). No significant change was observed with IL-2, IL-10, IL-6 or IL-8 with any of the nucleotides tested. These results show that ATPγS is able to induce the secretion of an overall pro-inflammatory cytokine environment from human lung tissue. ATPγS acts on P2Y2, 11, 12, 13 and P2X3 found on dendritic cells, suggesting that one of these receptors is involved in this process. The decrease in TNFα may be due to the involvement of a different cytokine secreting cell type. Further research into the specific cell types and P2 receptors involved in lung inflammation would help identify the true effects of nucleotides on the immune response.

Basoglu. O. K et al. 2005. Chest 128: 1905-9.

la Sala. A et al. G. 2003. Journal of Leukocyte Biology 73: 339-343