Burn-activated neutrophils and tumor necrosis factor-alpha alter endothelial cell actin cytoskeleton and enhance monolayer permeability

Background. This study examined the hypothesis that exposure of an endothel ial cell (EC) monolayer totumor necrosis factor-alpha (TNF-alpha) and that burn-activated neutrophils alter EC actin cytoskeleton and enhance the perm eability of the monolayer. Methods. Neutrophils were harvested from rats that had undergone a 45 % sur face area burn (BURN-neutrophil) or uninjured control rats. ECs were grown on polyester filters or fibronectin-coated glass slides and exposed for 4 h ours to media, TNF-alpha (100 ng/mL), or TNF-alpha plus BURN-neutrophil or uninjured control rats (10(7) cells). Monolayer permeability was assessed m easuring the flux of albumin across the cells. EC surface area and microfil ament number and length were determined by the staining of actin microfilam ents with rhodamine phalloidin followed by fluorescent microscopy. Results. The amount of albumin that moved across the monolayer in response to TNF-alpha plus BURN-neutrophil was twice that of media alone (P < .05) o r TNF-alpha alone (P < .05). The number and length of actin microfilaments in ECs exposed to TNF-alpha plus BURN-neutrophil were significantly less th an that of cells exposed to media alone or TNF-alpha alone. Conclusions. These data are consistent with a hypothesis that TNF-alpha plu s BURN-neutrophil affect endothelial monolayer permeability by altering EC actin cytoskeletal organization.