[48] Endothelial Culture, Neutrophil or Enzymic Generation of Free Radicals: In Vitro Methods for the Study of Endothelial Injury

In vitro culture of endothelial cells from a variety of anatomic sites and various species permits the study of the inflammatory response at a cellular level. The interactions of endothelial cells with polymorphonuclear leukocytes (PMN), particularly those activated by inflammatory mediators, such as certain complement components, seem critical to the vasodilatation and permeability alterations that characterize acute inflammation. The metabolic products of activated PMN, especially reactive oxygen species (ROS) and lysosomal proteases, contribute to the inflammatory response and have been implicated in many clinical syndromes. This chapter describes a method for determining toxic effects of PMN products upon endothelial cells in vitro. It uses cultured human umbilical vein endothelial cells that are mixed with activated PMN to induce release of endothelial-bound radiolabel and measures both cellular injury and detachment. Specific inhibitors may be used to determine the contribution of ROS (or other substances) produced by the neutrophil while PMN production of these products can be measured directly and correlated with the degree of endothelial injury. ROS may also be generated enzymically in the absence of PMN to further explore their effects upon the endothelial cells.