Accelerated internalization and detoxification of endotoxin by anti-lipopolysaccharide antibody is an Fc receptor-mediated process

Background. Interaction between lipopolysaccharide (LPS), LPS-binding protein, and the CD14 receptor at the surface of LPS-responsive cells results in inflammatory cytokine release and internalization and detoxification of LPS. Monoclonal antibodies (mAbs) raised against the deep-core lipid A or the O-linked polysaccharide moieties of LPS accelerate internalization and detoxification of LPS without stimulating cytokine release. This study was conducted to test the hypothesis that the antibody-mediated internalization of LPS is an Fc receptor (FcR)-mediated process. Methods. Fluoroisothiocyanate (FITC)-conjugated Escherichia coli O111:B4 LPS was incubated with RAW 264.7 cells and allowed to internalize for 2 hours in the presence and absence of anti-LPS, anti-CD14, and isotype control mAbs, and Fab fragments from the anti-CD14, anti-Fc receptor, and control mAbs. Tumor necrosis factor-alpha (TNF-alpha) release was measured by WEHI 164 cell bioassay. FITC-LPS uptake was measured by flow cytometry. Statistical analysis was by analysis of variance and Fisher exact test. Results. Addition of anti-LPS antibodies resulted in a 30- to 40-fold acceleration of LPS internalization (P<.01) in agreement with previous studies. This increase was blunted by anti-CD14 and also by isotype control holo-antibody (P<.01), but not by Fab fragments from anti-CD14 or isotype control antibody. Both anti-FcR antibodies and Fab fragments blocked anti-LPS antibody-stimulated uptake of FITC-LPS. Both intact anti-CD14 holo-antibody and Fab fragments blocked TNF-alpha release (P<.01). Conclusions. Clearance and detoxification of LPS are thought to be essential to the host response to endotoxin. It has been shown that antibodies to LPS accelerate its internalization by monocytic cell lines without increasing the elaboration of cytokines. We found that specific blockade of CD14 by Fab fragments could block TNF-<alpha> release but not alter the accelerated internalization of LPS produced by anti-LPS antibodies. In contrast, a nonspecific blockade of internalization was produced by competing antibody, which suggests a mechanistic role for the FcR. Specific blockade of FcR by either holo-antibody or Fab fragments blocked accelerated internalization, which confirms a FcR mechanism. We conclude that the accelerated internalization of LPS produced by anti-LPS antibody is an Fc receptor-media led process. These results have significance for the development of adjuvant immunotherapy for gram-negative bacterial sepsis.