Bacterial lipoproteins induce distinct IL-8 inductions relying on their lipid moieties in pulmonary epithelial cells

Pulmonary epithelial cells play a crucial role in host defense against bacterial insult. However, the innate immunity in pulmonary epithelium is relatively less characterized than that of professional immune cells. In the current study, we investigated IL-8 induction by bacterial cell wall components in human lung epithelial A549 cells and primary human bronchial epithelial cells. We found that lipoproteins are the most potent IL-8 inducers among bacterial cell wall components. Using synthetic lipopeptides that mimic bacterial lipoprotein pam2CSK4 and pam3CSK4, we further investigated signal transduction mechanism. Interestingly, IL-8 secretion appeared higher upon diacylated pam2CSK4 treatment than triacylated pam3CSK4. A role of TLR2 in lipopeptide-induced IL-8 secretion was monitored by siRNA transfection and functional blocking antibodies. We found that TLR2 in A549 cell localizes to intracellular compartment. Activation mechanism for the intracellular TLR2 differed between pam2CSK4 and pam3CSK4. Pam2CSK4 utilized a lipid-raft dependent mechanism while pam3CSK4 utilizes clathrin-dependnet endocytosis. Using CHO/CD14/TLR2 cell line, we found the distinct IL-8 induction level initiates at the receptor activation level. Subsequent pharmacological inhibitor studies showed that pam2CSK4 and pam3CSK4 utilizes different MAPK pathways. Downstream transcription factor activation was determined by electro-mobility shift assay (EMSA) and luciferase reporter assays. The results showed distinct pattern of activation for NF-{kappa}B, NF-IL6 and AP-1 from human IL-8 promoter. These results suggest that bacterial lipoproteins from cell walls are an important determinant in initiating tissue inflammation in pulmonary epithelium upon bacterial insult.