The evaluation of macrophages following bacterial or cytokine challenge

Phosphatidic acid (PA) has been shown to be involved in several pathophysiological processes, including cellular signaling and inflammation. The present study was conducted to evaluate the viability of RAW264.7 macrophages following treatment with lipopolysaccharides (LPS) or TNFa. Our hypothesis is that activation of the phosphatidylcholine-phospholipase D (PC-PLD) pathway leading to phosphatidic acid production plays a role in both cytokine and LPS signaling in macrophages. Results from this study show that RAW264.7 macrophages treated with LPS or TNFa did not affect cellular viability or cause increased cellular damage. This was evident by similar cellular viability results in treated and untreated cells as compared to the control. Cells treated with 2 microg/ml of LPS or 5 ng/ml of TNFa displayed similar changes in membrane phospholipids. PA formation in cells treated with LPS increased four-fold, compared to a two-fold increase in the TNFa treatment group. These increases in PA are likely related to the increase in cytokine production of both groups. DAG production increased nearly three-fold for both the LPS and TNFa treated groups. The addition of an inhibitor, D609, did not affect PA levels or cytokine production. Regardless to the stimuli used, cellular signaling leading to cytokine production was mediated via a common event, an increase in PA. Results from this study suggest that the PAPLD signaling pathway may promote inflammatory cytokine production.