In vitro analysis of pulmonary inflammation using rat lung organ cultures

We have analyzed leukocyte to lung adhesive interactions and neutrophil-mediated lung injury using a rat lung organ culture system. Rat lung slices were maintained in tissue culture on gelatin sponges floating at the gas-liquid interface. Maintenance of three-dimensional alveolar structure, critical to the viability of lung tissue, was achieved by instilling 0.5% agarose (in 37 degrees C RPMI 1640) into the lungs during tissue explanation. Quantitative neutrophil to lung adhesive interactions were examined using an adaptation of the Woodruff-Stamper frozen section binding assay. Pretreatment of organ cultures with recombinant human tumor necrosis factor (rhTNF) resulted in a protein synthesis-dependent three- to fourfold increase in adhesiveness for neutrophils. Time course and mononuclear leukocyte binding experiments revealed that TNF-induced rat lung adhesiveness peaks at 4 h and is largely neutrophil-specific. Agonist-induced activation of neutrophils in the presence of [3H]leucine-labeled organ cultures resulted in lung injury as assessed by radioisotope release. These observations are consistent with endothelial cell culture data that indicate that TNF-induced endothelium exhibits a protein synthesis-dependent increase in adhesiveness for neutrophils. These data validate rat lung organ cultures as a model system that can be used to assess mechanisms of neutrophil adhesion and leukocyte-mediated tissue injury.