The effect of phospholipase A2 on bacterial translocation in a cell culture model.
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.
Sawai
T
T
University of Michigan Medical School, Section of Pediatric Surgery, Mott Children's Hospital, Ann Arbor 48109-0245, USA.
Usui
N
N
Dwaihy
J
J
Drongowski
R A
RA
Abe
A
A
Coran
A G
AG
Harmon
C M
CM
D018988:Bacterial Translocation / Q000502:physiology*; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D017097:Electric Impedance; D020895:Enterocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010741:Phospholipases A / Q000502:physiology*; D054467:Phospholipases A2
D010741:Phospholipases A; D054467:Phospholipases A2