The effect of phospholipase A(2) on bacterial translocation in a cell culture model

The activity of phospholipase (PL)A(2) is elevated in the intestinal epithe lia of patients with inflammatory bowel disease (IBD). Recently, we reporte d that lysophosphatidylcholine (L-PC), the PLA(2) hydrolysis product of pho sphatidylcholine (PC), stimulates bacterial translocation (BT) in an entero cyte cell-culture model. These two observations stimulated us to examine th e effects of extracellular PLA(2) on intestinal epithelial permeability. Hu man Caco-2 enterocytes were grown to confluence on porous filters in the ap ical chamber of a two-chamber cell-culture system. Monolayer integrity and tight-junction permeability were measured by dextran blue (DB) permeability and transepithelial electric resistance (TEER). Monolayers were treated wi th PC, LPC, or PLA(2) with and without PC. The magnitude of BT was determin ed 2 h after treatment by adding Escherichia coli to the apical chamber fol lowed by quantitatively culturing basal chamber samples. Thin-layer chromat ography (TLC) was utilized to verify PLA(2) hydrolysis of PC to L-PC. Stati stical analysis was performed by one-way analysis of variance. The magnitud e of BT across monolayers pretreated with PLA(2) + PC significantly increas ed compared to either PC or PLA(2) (6.83 +/- 0.069, 2.41 +/- 0.46, and 3.06 +/- 1.14 log10 colony forming units/ml, respectively, P < 0.05). Absence o f DB-permeability in any group confirmed monolayer integrity. TLC of PL sam ples harvested from the apical monolayer surface confirmed PC hydrolysis. P LA(2) mediates hydrolysis of PC to L-PC when both are applied to the apical surface of cultured enterocyte monolayers, resulting in increased BT and i ncreased TEER with no damage to monolayer integrity. These observations may have implications in the pathogenesis and treatment strategies for IBD.