Influence of antiseptic agents on interleukin-8 release and transmigration of polymorphonuclear neutrophils in a human in vitro model of peritonitis

Abstract

Background: The aim of this study was to evaluate the influence of taurolidine (TAU) and polyhexanid (POLY) on basic inflammatory reactions during peritonitis by using an in vitro model of human peritoneum.

Materials and methods: Human umbilical vein endothelial cells (HUVEC) and human peritoneal mesothelial cells (HPMC; concentration: 2x10(5)/cm2) were brought on a collagen-coated filter insert with 3-microm pore size (HUVEC on the bottom, HPMC on the top), thus resulting in a two-chamber peritoneal model. After 5 days, confluence of the cells was reached, and HPMC were stimulated with 0.5 mL of TNF-alpha (10 microg/mL) for 4 h. Afterwards, 0.5 mL of TAU (1% and 2%) or 0.5 mL of POLY (0.1% and 0.2%) solution were added to the upper (HPMC) compartment. Polymorphonuclear neutrophils (PMN, 10(6)/mL) were placed in the lower compartment 1 h later. After 2 and 6 h, aliquots were taken from the upper compartment and transmigrated PMN were counted. Interleukin-8 (IL-8) concentrations were measured in both compartments by chemiluminescent enzyme immunometric assay. Expression of the adhesion molecules P-selectin and intercellular adhesion molecule-1 (ICAM-1) was assessed by immunohistochemistry. Controls were either TNF-alpha-stimulated HPMC without any antiseptic agents, or stimulated HPMC where TNF-alpha had been substituted by culture medium. Each experiment was performed in triplicate.

Results: Stimulation with TNF-alpha led to a time-dependent increase of IL-8 secretion to the apical compartment resulting in a gradient between both chambers, as well as to a time-dependent increase of PMN transmigration and expression of adhesion molecules. IL-8 gradients and PMN migration were significantly higher as compared to the other groups (p<0.05). After substitution of the stimulus by culture medium, significantly less IL-8 was measured in both compartments. PMN transmigration was almost absent (p<0.05). Addition of POLY and TAU led to comparable low IL-8 gradients with concomitant low PMN transmigration. The initially detected expression of adhesion molecules significantly decreased during the observation time. The IL-8 gradient in all groups correlated significantly with PMN transmigration (r=0.74226; p<0.0001).

Conclusion: The diminished IL-8 response together with low PMN transmigration rates after addition of TAU and POLY may reflect either antiinflammatory effects or cellular damage.