The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures

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Abstract

Introduction: Lipopolysaccharide (LPS) contamination of commercially available proteins has seriously impeded research on citrullinated fibrinogen (cit-Fb) in rheumatoid synovial cells (RSCs). Methods: RSCs obtained from 4 rheumatoid arthritis patients who underwent full knee arthroplasty were cultured, stimulated with cit-Fb, and cytokine expression levels were measured. We then evaluated polymyxin-B (PMB), heat inactivation, and rough (R)-type LPS mutants for rapid detection of LPS contamination. Results: cit-Fb induced expression of CXCL10 and IFNB in RSCs via the toll-like receptor. PMB inhibited cit-Fb-mediated CXCL10 gene expression but not protein expression induced by 20 μg/mL cit-Fb. Heat inactivation did not affect LPS-mediated CXCL10 or IL-6 induction; however, cit-Fb-mediated CXCL10expression was inhibited. Wild-type LPS from Escherichia coli (WT-LPS) strongly induces CXCL10 expression, but induction by Ra-LPS was weak, and induction by Rc- and Re-LPS was minimal. Re-LPS suppression of WT-LPS-mediated CXCL10 induction in RSCs and peripheral blood monocytes (PBMs) was dose dependent. Furthermore, Re-LPS completely suppressed cit-Fb-mediated CXCL10 induction in RSCs and PBMs. Conclusion: To easily identify LPS contamination during routine experiments, our results suggest that Re-LPS is a better tool for rapid detection of LPS contamination compared to PMB and heat treatment.

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Most cited references 34

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Lipopolysaccharide endotoxins.

Christian Raetz, Chris Whitfield (2002)

Bacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating "core" oligosaccharide, and a distal polysaccharide (or O-antigen). Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens. Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics. Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist. Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection.

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Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages.

Baochong Gao, Min-Fu Tsan (2003)

Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor alpha (TNFalpha) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70-1) with a LPS content of 1.4 pg/microg was unable to induce TNFalpha release by RAW264.7 murine macrophages at concentrations up to 5 microg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70-2) at 1 microg/ml with a LPS content of 0.2 ng/microg was able to induce TNFalpha release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70-1 to induce TNFalpha release was not because of defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70-2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFalpha-inducing activity of rhHsp70-2. The addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNFalpha-inducing activity as observed with rhHsp70-2. The TNFalpha-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70-1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFalpha release from murine macrophages and that the observed TNFalpha-inducing activity in the rhHsp70-2 preparation is entirely due to the contaminating LPS.

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Endotoxin Contamination in Nanomaterials Leads to the Misinterpretation of Immunosafety Results

Yang Li, Mayumi Fujita, Diana Boraschi (2017)

Given the presence of engineered nanomaterials in consumers’ products and their application in nanomedicine, nanosafety assessment is becoming increasingly important. In particular, immunosafety aspects are being actively investigated. In nanomaterial immunosafety testing strategies, it is important to consider that nanomaterials and nanoparticles are very easy to become contaminated with endotoxin, which is a widespread contaminant coming from the Gram-negative bacterial cell membrane. Because of the potent inflammatory activity of endotoxin, contaminated nanomaterials can show inflammatory/toxic effects due to endotoxin, which may mask or misidentify the real biological effects (or lack thereof) of nanomaterials. Therefore, before running immunosafety assays, either in vitro or in vivo, the presence of endotoxin in nanomaterials must be evaluated. This calls for using appropriate assays with proper controls, because many nanomaterials interfere at various levels with the commercially available endotoxin detection methods. This also underlines the need to develop robust and bespoke strategies for endotoxin evaluation in nanomaterials.

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Journal

Journal ID (publisher-id): PAT

Journal ID (nlm-ta): Pathobiology

Journal ID (doi): 10.1159/issn.1015-2008

Title: Pathobiology

Publisher: S. Karger AG

ISSN (Print): 1015-2008

ISSN (Electronic): 1423-0291

Publication date Subscription-year: 2022

Publication date (Print): March 2022

Publication date (Electronic): 19 November 2021

Volume: 89

Issue: 2

Pages: 92-100

Affiliations

[_a] _aDepartment of Clinical Immunology and Rheumatology, Hiroshima University Hospital, Hiroshima, Japan

[_b] _bDepartment of Periodontal Medicine, Hiroshima University, Hiroshima, Japan

[_c] _cDepartment of Immunology, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan

[_d] _dThe First Department of Internal Medicine, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan

Author information

Hiroki Kohno https://orcid.org/0000-0003-2947-6216

Shintaro Hirata https://orcid.org/0000-0002-2474-9943

Article

Publisher ID: 520022 Other ID: Pathobiology 2022;89:92–100

DOI: 10.1159/000520022

PubMed ID: 34802006

SO-VID: 091d1ed7-d36c-4675-ad56-9bcca9b49199

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Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

History

Date received : 16 June 2021

Date accepted : 02 October 2021

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Figures: 3, Pages: 9

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The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures

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Karger: Oncology

Most cited references 34

Lipopolysaccharide endotoxins.

Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages.

Endotoxin Contamination in Nanomaterials Leads to the Misinterpretation of Immunosafety Results

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