Evidence for lipopolysaccharide as the predominant proinflammatory mediator in supernatants of antibiotic-treated bacteria

Lipopolysaccharide (LPS), purified from gram-negative bacteria, is well known to induce proinflammatory responses in monocytes and macrophages, and release of LPS from the microbial surface has been suggested to be an important initiating event in the sepsis syndrome. However, numerous studies have documented that a variety of constituents present in the outer cell membrane of gram-negative bacteria have the capacity to activate cells of the immune system. Given that the majority of immunotherapeutic approaches designed to intervene in gram-negative sepsis have to date targeted the LPS molecule, it would be of value to assess the relative proinflammatory properties of LPS and other gram-negative structures. Experiments were therefore undertaken to assess stimulation of human monocytes by components released from Escherichia coli following bacteriolysis by the cell wall-active antibiotic ceftazidime. As assessed by both induction of procoagulant activity and release of tumor necrosis factor, bacterial culture supernatants contain significant proinflammatory activity. When culture supernatants are fractionated via either velocity sedimentation in sucrose gradients or isopycnic density gradient ultracentrifugation in cesium chloride, the predominant monocyte-stimulating activity is identified in LPS-containing fractions. Further, such activity can be readily abrogated by the addition of polymyxin B. These results provide support for the hypothesis that LPS may be responsible for the majority of the proinflammatory activity released from E. coli following bacteriolysis in vitro.