Lipopolysaccharide Clearance Research Articles

Human Anti-Endotoxin Antibody HA-1A Mediates Complement-Dependent Binding of Escherichia coli J5 Lipopolysaccharide to Complement Receptor Type 1 of Human Erythrocytes and Neutrophils

HA-1A has been shown clinically to decrease mortality in septic patients with gram-negative bacteremia. In this study, the ability of HA-1A to augment the serum complement-dependent immune adherence of 125I-labeled Escherichia coli J5 lipopolysaccharide (LPS) to human erythrocytes (RBC) and polymorphonuclear leukocytes (PMNL) was evaluated. In vitro studies indicated three things: HA-1A mediates immune adherence of 125I-J5 LPS to human RBC and PMNL in a dose-dependent manner; under these conditions, high concentrations of LPS (400 ng/mL) could be specifically bound. Immune adherence occurs via the classical complement pathway as demonstrated by its calcium dependence; HA-1A-J5 LPS-C' immune complexes bound to CR1 on human RBC and PMNL. PMNL binding and internalization of immune complexes was demonstrated by trypsin stripping of externally bound immune complexes. These studies support the proposal that HA-1A can lower the bioavailability of endotoxin by mediating binding and potential clearance of LPS via human RBC through the reticuloendothelial system or via direct internalization by peripheral blood PMNL.

Ultrastructural and Immunocytochemical Study of the Uptake and Distribution of Bacterial Lipopolysaccharide in Human Monocytes

Interaction of bacterial lipopolysaccharide (LPS) with monocytes stimulates production of a variety of mediators that are involved in the pathogenesis of septic shock and wound repair. We report here the mechanisms of LPS uptake and intracellular distribution of LPS in human monocytes. Ficoll-Hypaque-purified peripheral mononuclear cells (PBMC) were exposed to LPS from rough Escherichia coli (J5) or to biotin-conjugated LPS (biotin-LPS) from smooth E. coli (0111:B4), or to fluorescein isothiocyanate-conjugated LPS of E. coli (055:B5) at 37 degrees C for various times and processed for electron microscopy, immunocytochemistry, and flow cytometry. Monocytes were identified by the presence of numerous cytoplasmic peroxidase-positive granules or by monoclonal antibodies against monocyte. LPS micelles were identified by their specific bilayer structure, staining of horseradish peroxidase reaction product, or colloidal gold using biotin-LPS or a monoclonal antibody to LPS. Binding of LPS to cell surface was observed 5 min after incubation with LPS. Intracellular localization of LPS micelles was found 30 min following exposure to LPS. Prolonged incubation with LPS increased intracellular LPS. Intracellularly, LPS micelles were found in large membrane-bound vacuoles, in small vesicles, and in the cytoplasm and nucleus. They were also observed in association with the cytomembrane of various organelles. The overall results indicate that LPS may be taken up by monocytes by direct passive diffusion through ruptures of plasma membrane, pinocytosis, and phagocytosis, involving specific and/or nonspecific binding, and suggest that peripheral blood monocytes play an important role in clearance of LPS; that LPS may have broad effects on cell functions; and that the nonspecific binding to various cytomembranes may be destructive to cell organelles and cells in general.

Enhancement of cell attachment of a substrate coated with oral bacterial endotoxin by plasma fibronectin.

Normal human fibroblasts were inhibited from attaching to a culture surface that was coated with endotoxic lipopolysaccharide (LPS) isolated from oral strains of Bacteroides or Fusobacterlum. The LPS from Fusobacterium inhibited cell attachment to a greater degree (p < 0.001) than that of the LPS from Bacteroides, and at a concentration as low as 12.5 ng/cm2 (p < 0.001), When LPS‐coated culture dishes (12.5 ng/cm2) were incubated with concentrations of plasma fibronectin as low as 50 μg/ml for at least 15 min, the fibronectin significantly enhanced cell attachment, returning it to control levels (p < 0.001). Futhermore, when plasma fibronectin (50 μg/ml) was compared to other agents such as bile acids, citrated serum, and human plasma fraction IV1 (Cohn), which have been shown to affect the toxicity of LPS, fibronectin led to significantly more cell attachment in the presence of LPS than any other treatment (p < 0.001). Evidence is presented by means of transmission electron microscopy (TEM) that LPS may bind to fibronectin. Subsequently, the cell surface interacts with the fibronectin‐LPS and internalizes it via phagocytosis. This mechanism provides for the clearance of LPS from the culture surface.