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

Background. This study examined the hypothesis that exposure of an endothelial cell (EC) monolayer to tumor necrosis factor-α (TNF-α) and that burn-activated neutrophils alter EC actin cytoskeleton and enhance the permeability of the monolayer. Methods. Neutrophils were harvested from rats that had undergone a 45 % surface area burn (BURN-neutrophil) or uninjured control rats. ECs were grown on polyester filters or fibronectin-coated glass slides and exposed for 4 hours to media, TNF-α (100 ng/mL), or TNF-α plus BURN-neutrophil or uninjured control rats (10⁷ cells). Monolayer permeability was assessed by measuring the flux of albumin across the cells. EC surface area and microfilament number and length were determined by the staining of actin microfilaments with rhodamine phalloidin followed by fluorescent microscopy. Results. The amount of albumin that moved across the monolayer in response to TNF-α plus BURN-neutrophil was twice that of media alone (P < .05) or TNF-α alone (P < .05). The number and length of actin microfilaments in ECs exposed to TNF-α plus BURN-neutrophil were significantly less than that of cells exposed to media alone or TNF-α alone. Conclusions. These data are consistent with a hypothesis that TNF-α plus BURN-neutrophil affect endothelial monolayer permeability by altering EC actin cytoskeletal organization.