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Defensins reduce the barrier integrity of a cultured epithelial monolayer without cytotoxicity
S D Nygaard1, T Ganz, M W Peterson
1Department of Internal Medicine, University of Iowa, Iowa City.
Abstract:
Polymorphonuclear leukocytes (PMN) contribute to epithelial injury at sites of inflammation, but their mechanisms of action are incompletely understood. PMN can injure target tissues by oxidative and nonoxidative mechanisms. Included in the nonoxidative mechanisms are defensins (DEF), small (3.5 to 4.0 kD), arginine- and cysteine-rich polypeptides. DEF are bactericidal, fungicidal, viricidal, and tumoricidal, but their ability to contribute to inflammatory injury has not been extensively evaluated. One marker of inflammatory injury is disrupted epithelial barrier integrity. Using Madin-Darby canine kidney (MDCK) epithelial monolayers, we measured the effect of both human and rabbit DEF on barrier integrity using mannitol permeability (Pmann) and transepithelial electrical resistance (Rt). Human DEF (HNP1-3, 2:2:1 molar ratio) increased Pmann in a time- and concentration-dependent manner and Rt fell progressively over a 48-h period after exposure of monolayers to HNP1-3. Rabbit DEF peptide 1 (NP-1) also increased Pmann, but rabbit peptide 5 (NP-5) had no effect on Pmann. To investigate the role of charge, HNP1-3 was added to the monolayers with the polyanions heparin or sulfated dextran. Heparin and sulfated dextran only partially inhibited the increase in Pmann. Fetal bovine serum (FBS), however, completely inhibited the effect of HNP1-3, but this protection was only partially explained by the anionic protein, albumin. The FBS protection was time dependent and was present when FBS was added up to 16 h after exposure to HNP1-3. While both HNP1-3 and NP-1 increased epithelial permeability, neither were cytolytic to MDCK cells as measured by lactate dehydrogenase release.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Defensins (DEF), antimicrobial peptides, disrupt epithelial barrier integrity by increasing permeability. Fetal bovine serum (FBS) protects against this DEF-induced injury, highlighting a potential therapeutic mechanism.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMN) contribute to inflammatory epithelial injury through oxidative and nonoxidative mechanisms.
- Defensins (DEF), a class of antimicrobial peptides, are involved in nonoxidative PMN-mediated injury.
- The role of DEF in inflammatory injury, particularly in disrupting epithelial barrier integrity, requires further investigation.
Purpose of the Study:
- To evaluate the effect of human and rabbit defensins on epithelial barrier integrity.
- To investigate the mechanisms by which defensins impact epithelial permeability and resistance.
- To identify factors that can protect epithelial monolayers from defensin-induced damage.
Main Methods:
- Utilized Madin-Darby canine kidney (MDCK) epithelial monolayers to assess barrier integrity.
- Measured mannitol permeability (Pmann) and transepithelial electrical resistance (Rt) to quantify barrier function.
- Assessed the effects of human defensins (HNP1-3) and rabbit defensins (NP-1, NP-5), with and without polyanions and fetal bovine serum (FBS).
Main Results:
- Human defensins (HNP1-3) and rabbit defensin NP-1 increased epithelial permeability (Pmann) and decreased resistance (Rt) in a time- and concentration-dependent manner.
- Rabbit defensin NP-5 had no significant effect on epithelial permeability.
- Fetal bovine serum (FBS) completely inhibited the HNP1-3-induced increase in permeability, with protection observed even when added up to 16 hours post-exposure.
Conclusions:
- Defensins can disrupt epithelial barrier integrity, contributing to inflammatory injury.
- Epithelial cells are not rendered cytolytic by defensin exposure.
- Fetal bovine serum offers significant protection against defensin-mediated epithelial barrier disruption, suggesting a potential therapeutic role.