Exogenous sphingomyelinase causes impaired intestinal epithelial barrier function.
OBJECTIVE
To test the hypothesis that hydrolysis of sphingomyelin to ceramide changes the composition of tight junctions (TJs) with increasing permeability of the intestinal epithelium.


METHODS
Monolayers of Caco-2 cells were used as an in vitro model for the intestinal barrier. Permeability was determined by quantification of transepithelial flux and transepithelial resistance. Sphingolipid-rich membrane microdomains were isolated by a discontinuous sucrose gradient and characterized by Western-blot. Lipid content of microdomains was analysed by tandem mass spectrometry. Ceramide was subcellularly localized by immunofluorescent staining.


RESULTS
Exogenous sphingomyelinase increased transepithelial permeability and decreased transepithelial resistance at concentrations as low as 0.01 U/mL. Lipid analysis showed rapid accumulation of ceramide in the membrane fractions containing occludin and claudin-4, representing TJs. In these fractions we observed a concomitant decrease of sphingomyelin and cholesterol with increasing concentrations of ceramide. Immunofluorescent staining confirmed clustering of ceramide at the sites of cell-cell contacts. Neutralization of surface ceramide prevented the permeability-increase induced by platelet activating factor.


CONCLUSIONS
Our findings indicate that changes in lipid composition of TJs impair epithelial barrier functions. Generation of ceramide by sphingomyelinases might contribute to disturbed barrier function seen in diseases such as inflammatory, infectious, toxic or radiogenic bowel disease.
Bock
Jurgen
J
Department of Internal Medicine I, University of Regensburg, Regensburg, Germany. juergen.bock@klinik.uni-regensburg.de
Liebisch
Gerhard
G
Schweimer
Joachim
J
Schmitz
Gerd
G
Rogler
Gerhard
G
D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D002518:Ceramides / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D062506:Claudin-4; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000473:pathology* / Q000503:physiopathology; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin; D010972:Platelet Activating Factor / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*; D013109:Sphingomyelins / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
C568157:CLDN4 protein, human; D002518:Ceramides; D062506:Claudin-4; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010972:Platelet Activating Factor; D013109:Sphingomyelins; D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase