Background
Leukotrienes are pro-inflammatory lipid mediators, derived from arachidonic acid via 5-lipoxygenase (5-LO). Leukotriene B4 (LTB4) is an effective neutrophil (PMN) chemoattractant, as well as being a major product of PMN priming. LTB4 is rapidly metabolized into products that are thought to be inactive, and little is known about the effects of LTB4 on the pulmonary endothelium. We hypothesize that LTB4 and its metabolites are effective PMN priming agents and cause pro-inflammatory activation of pulmonary endothelial cells.
Methods
Isolated PMNs were primed (5 min, 37°C) with serial concentrations 10−11–10−5M of LTB4 and its metabolites: 6-trans-LTB4, 20-OH-LTB4, and 20-COOH-LTB4 and then activated with fMLP. Primary human pulmonary microvascular endothelial cells (HMVECs) were incubated with these lipids (6 hrs, 37°C, 5% CO2) and intercellular adhesion molecule-1 (ICAM-1) was measured by flow cytometry. PMN adhesion was measured by myeloperoxidase assays and to ensure that these reactions were specific to the LTB4 receptors BLT1 and BLT2 were antagonized with CP105,696 (BLT1) or silenced with siRNA (BLT1 and BLT2).
Results
LTB4 and its metabolites primed PMNs over a wide range of concentrations depending upon the specific metabolite. In addition, at high concentrations these lipids also caused increases in the surface expression of ICAM-1 on HMVECs and induced HMVEC-mediated adhesion of PMNs. Silencing of BLT2 abrogated HMVEC activation and blockade of BLT1 inhibited the observed PMN priming activity. We conclude that LTB4 and its ω-oxidation and non-enzymatic metabolites prime PMNs over a range of concentrations and activate HMVECs. These data have expanded the repertoire of causative agents in ALI and post-injury multiple organ failure.