Influence of synthetic antiendotoxin peptides on lipopolysaccharide (LPS) recognition and LPS-induced proinflammatory cytokine responses by cells expressing membrane-bound CD14.
Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety. Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential. The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), as measured by bioassays. PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells. The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergized with PMB and SAEP-4. LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic. LPS-induced TNF-alpha release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb. SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum. Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells. Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.
Iwagaki
A
A
Department of Medicine, Uniformed Services University of the Health Sciences, F. Edward Hebert School of Medicine, Bethesda, Maryland 20814, USA.
Porro
M
M
Pollack
M
M
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D016207:Cytokines / Q000096:biosynthesis*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000502:physiology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011112:Polymyxin B / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D009842:Oligopeptides; D010455:Peptides; D010456:Peptides, Cyclic; C405071:SAEP-2 peptide; C405072:SAEP-4 peptide; D011112:Polymyxin B