ANTIENDOTOXIN MONOCLONAL-ANTIBODIES INHIBIT SECRETION OF TUMOR-NECROSIS-FACTOR-ALPHA BY 2 DISTINCT MECHANISMS

Objective To determine whether monoclonal antibodies (mAbs) directed a gainst lipopolysaccharide (LPS, endotoxin) act by promoting LPS neutra lization, LPS uptake by macrophages, or both processes, the authors as sessed the effects of these agents on LPS-induced cytokine secretion a nd cellular uptake of LPS. Summary Background Data MAbs directed again st LPS have been shown to attenuate LPS-induced macrophage tumor necro sis factor-alpha (TNF-alpha) secretion, a process that may contribute to protective capacity. The mechanisms by which this process occurs ha ve not been established. Methods MAbs directed against LPS were evalua ted in vitro for their capacity to (1) inhibit TNF-alpha secretion, an d (2) alter fluorescein isothiocyanate-labeled LPS uptake (employing f low cytometry analysis and fluorescence microscopy) by the macrophage- like cell line RAW 264.7. Results MAb 8G9, an IgG3 directed against th e O-antigen polysaccharide region of Escherichia coli 0111:B4 LPS, sig nificantly reduced LPS-induced TNF-alpha secretion and promoted a more than 40-fold increase in LPS uptake by macrophages. The authors estab lished that this was mediated by a Fc receptor-mediated process becaus e 8G9 F(ab')2 fragments that lack the Fc portion of the IgG molecule w ere capable of inhibiting TNF-alpha secretion, but did not promote inc reased LPS uptake to the same degree. Cross-reactive, anti-deep core/l ipid A mAb 1B6, an IgG2a, also promoted uptake of E. coli 0111:B4 LPS and O-antigen polysaccharide-deficient E. coli J5 LPS, but only inhibi ted TNF-alpha secretion induced by E. coli J5 LPS to which it binds mo st efficiently. MAb 3D10, an IgM also directed against the O-antigen p olysaccharide region of E. coli 0111:B4 LPS, inhibited TNF-alpha secre tion but did not increase cellular uptake of LPS, presumably acting so lely due to LPS neutralization. Polymyxin B, an antibiotic that binds stoichiometrically to the lipid A portion of LPS, inhibited TNF-alpha secretion and prevented cellular LPS uptake, Conclusions These results suggest that IgG and IgM anti-LPS mAbs exert protective capacity by e xtracellular neutralization of LPS, while IgG Fc receptor-mediated cel lular uptake also may serve to bypass macrophage activation and TNF-al pha secretion by promoting internalization and intracellular neutraliz ation.