DEFENSINS REDUCE THE BARRIER INTEGRITY OF A CULTURED EPITHELIAL MONOLAYER WITHOUT CYTOTOXICITY

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 nonoxidati ve 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 the ir ability to contribute to inflammatory injury has not been extensive ly evaluated. One marker of inflammatory injury is disrupted epithelia l barrier integrity. Using Madin-Darby canine kidney (MDCK) epithelial monolayers, we measured the effect of both human and rabbit DEF on ba rrier integrity using mannitol permeability (P(mann)) and transepithel ial electrical resistance (R(t)). Human DEF (HNP1-3, 2:2:1 molar ratio ) increased P(mann) in a time- and concentration-dependent manner and R(t) fell progressively over a 48-h period after exposure of monolayer s to HNP1-3. Rabbit DEF peptide 1 (NP-1) also increased P(mann), but r abbit peptide 5 (NP-5) had no effect on P(mann). To investigate the ro le of charge, HNP1-3 was added to the monolayers with the polyanions h eparin or sulfated dextran. Heparin and sulfated dextran only partiall y inhibited the increase in P(mann). Fetal bovine serum (FBS), however , completely inhibited the effect of HNP1-3, but this protection was o nly partially explained by the anionic protein, albumin. The FBS prote ction 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 epithe lial permeability, neither were cytolytic to MDCK cells as measured by lactate dehydrogenase release. These studies demonstrate that HNP1-3 and NP-1 alter the barrier integrity of a cultured epithelium without causing cytolytic injury and expand the possible contribution of DEF t o inflammatory injury.