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Intact rough‐ and smooth‐form lipopolysaccharides from E scherichia coli separated by preparative gel electrophoresis exhibit differential biologic activity in human macrophages
Author(s) -
Pupo Elder,
Lindner Buko,
Brade Helmut,
Schromm Andra B.
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12104
Subject(s) - lipopolysaccharide , chemistry , tlr2 , in vitro , innate immune system , tumor necrosis factor alpha , biological activity , escherichia coli , microbiology and biotechnology , receptor , biochemistry , chromatography , biology , immunology , gene
We established a new preparative separation procedure, based on DOC/ PAGE , to isolate intact lipopolysaccharide ( LPS ) fractions from natural LPS preparations of E scherichia coli . Analysis of the chemical integrity of LPS fractions by MS showed that no significant chemical modifications were introduced by the procedure. Contamination with toll‐like receptor 2 ( TLR 2)‐reactive cell‐wall components present in the natural LPS mixture was effectively removed by the procedure, as determined by the absence of reactivity of the purified fractions in a HEK 293‐ TLR 2 cell line. Biologic analysis of LPS fractions derived from E . coli O 111 in human macrophages demonstrated that the rough ( R ), semirough ( SR ) and smooth ( S ) LPS fractions were highly active at inducing tumor necrosis factor‐alpha ( TNF ‐α) in the presence of human serum; however, on a weight basis the R ‐ LPS and SR ‐ LPS fractions were more active, by a factor of 10–100, than was the S ‐ LPS fraction. Under serum‐free conditions, the natural LPS mixture, as well as the R ‐ LPS and SR ‐ LPS fractions, showed dose‐dependent activation of macrophages, although the response was attenuated by about 10‐ to 100‐fold. In contrast, the S ‐ LPS fraction failed to induce TNF ‐α. Remarkably, the dose–response of the natural LPS mixture resembled that of the R‐ LPS and SR‐ LPS fractions, supporting that short‐chain (R and SR) forms of LPS dominate the innate immune response of human macrophages to LPS in vitro . Biologic activity to the S‐ LPS fraction under serum‐free conditions could be restored by the addition of recombinant lipopolysaccharide‐binding protein ( LBP ). In contrast, soluble cluster of differentiation antigen 14 was not able to confer activity of the S‐ LPS fraction, indicating a crucial role of LBP in the recognition of S‐ LPS by human macrophages.