| The lipid A region of gram-negative bacterial lipopolysaccharide (LPS, endotoxin) binds to the macrophage cell surface and induces the production of a wide array of cytokines, including tumor necrosis factor-{dollar}alpha{dollar} (TNF-{dollar}alpha{dollar}), interleukin-1 (IL-1), and interleukin-6 (IL-6). Increasing evidence suggests that excessive secretion of these cytokines by host macrophages contributes to organ failure and death associated with serious gram-negative bacterial infections.; The aim of this project was to attempt to characterize macrophage-lipopolysaccharide interactions at the molecular and cellular levels using endotoxin antagonists. Two separate strategies were pursued to reach that aim. First, synthetic peptides derived from three different LPS binding proteins, Bactericidal permeability increasing protein (BPI), Limulus anti-LPS factor (LALF), and LPS binding protein (LBP), were studied for their ability to (a) bind to LPS and inhibit LPS-induced macrophage secretion of TNF-{dollar}alpha{dollar} and (b) to mediate bacteriolysis of gram-negative bacterial pathogens. Second, an anti-idiotypic monoclonal antibody designed to mimic lipid A was studied for its ability to (a) bind to the macrophage cell surface and (b) inhibit LPS-induced macrophage secretion of TNF-{dollar}alpha{dollar}.; The data provided in these studies indicated that the LPS-binding domain derived from BPI, LALF, and LBP represented an important structural motif utilized by LPS-binding proteins. In addition, studies utilizing an anti-idiotypic monoclonal antibody designed to mimic lipid A demonstrated that this antibody bound to the macrophage cell surface and inhibited LPS-induced macrophage secretion of TNF-{dollar}alpha{dollar}. Interpretation of these data together suggested that the cell surface molecule to which this anti-idiotypic monoclonal antibody bound was distinct from CD14 and suggested that there may be a macrophage cell surface protein expressing the amphipathic hairpin loop observed in the LPS-binding regions of BPI, LALF, and LBP. Localization of this structural motif to a specific macrophage cell surface protein may help to identify cell surface receptors other than CD14 that mediate LPS-induced cytokine secretion and may lead to the development of effective adjuvant therapies for the treatment of patients with gram-negative bacterial sepsis. |
