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

Abstract

The activity of phospholipase (PL)A2 is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD). Recently, we reported that lysophosphatidylcholine (L-PC), the PLA2 hydrolysis product of phosphatidylcholine (PC), stimulates bacterial translocation (BT) in an enterocyte cell-culture model. These two observations stimulated us to examine the effects of extracellular PLA2 on intestinal epithelial permeability. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical 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 with PC, L-PC, or PLA2 with and without PC. The magnitude of BT was determined 2 h after treatment by adding Escherichia coli to the apical chamber followed by quantitatively culturing basal chamber samples. Thin-layer chromatography (TLC) was utilized to verify PLA2 hydrolysis of PC to L-PC. Statistical analysis was performed by one-way analysis of variance. The magnitude of BT across monolayers pretreated with PLA2 + PC significantly increased compared to either PC or PLA2 (6.83 +/- 0.069, 2.41 +/- 0.46, and 3.06 +/- 1.14 log10 colony forming units/ml, respectively, P < 0.05). Absence of DB-permeability in any group confirmed monolayer integrity. TLC of PL samples harvested from the apical monolayer surface confirmed PC hydrolysis. PLA2 mediates hydrolysis of PC to L-PC when both are applied to the apical surface of cultured enterocyte monolayers, resulting in increased BT and increased TEER with no damage to monolayer integrity. These observations may have implications in the pathogenesis and treatment strategies for IBD.

Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers

“…PLC and PKC have both been implicated in the reversible melittin response, with PLA2 and cyclooxygenase-2 inhibitors only having effects after extended periods of time (>3 h). PLA2 activation has been shown to indirectly decrease TER of Caco-2 monolayers (41). While further studies are needed to elucidate a mechanism, it is likely that these prostaglandins play some role in the melittin effects on tight junctions.…”

“…It has been previously been shown that melittin is an activator of PLA2, (40) which resulted in an increase in electrogenic chloride secretion across rat colonic epithelium (24). Activation of PLA2 has been shown to decrease TER of Caco-2 cells via hydrolysis of phosphatidylcholine (41). A number of inhibitors of reported melittin activities were screened to assess the mechanism of action of the peptide on tight junctions.…”

Melittin as a Permeability Enhancer II: In Vitro Investigations in Human Mucus Secreting Intestinal Monolayers and Rat Colonic Mucosae

“…Low concentrations of melittin (0.17-0.7 mM) increased Isc due to the peptide_s PLA2 activation, with resultant prostaglandin E2 production. PLA2 is also linked with absorption enhancement, its activation mediates the hydrolysis of phosphatidylcholine to lysophosphatidylcholine which decreased TER in Caco-2 monolayers (35), suggesting that the concurrent permeation enhancing and ion secretory effects of melittin may involve PLA2. Quinacrine, an inhibitor of PLA2, had little effect on apical melittin-induced alterations in barrier function or Isc, suggesting a limited role for PLA2.…”

“…These include signalling molecules such as protein kinase C (PKC) (37), phospholipase A2 (PLA2) (35) and calcium dependant calmodulin (38). These aspects of melittin_s activity and its ability to disrupt the integrity of the cell membrane may be involved in the peptide_s cytotoxicity which is considered a limiting factor in the use of melittin in infectious disease (20).…”

“…In addition the loop diuretic, bumetanide significantly reduced the extent of melittin_s apical induced secretory response (Table II). Melittin has been reported as an agonist of phospholipase A2 (35). However, the phospholipase A2 (PLA 2 ) inhibitor, quinacrine (10 mM) had no significant effect on melittininduced decreases in TER or on the induced increase in ion secretion/absorption (Table II).…”