CoChat
Sign Up
Cited by 396Heat shock proteins researchImmune Response and InflammationProtein purification and stability

Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor α Release by Murine Macrophages

Open access
Gao, Baochong, Tsan, Min-Fu·2003·Journal of Biological Chemistry
DOI 10.1074/jbc.m208742200
Open this paper in CoChat — free account

Abstract

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.

Integrity signals

Registry

Crossref · 10.1074/jbc.m208742200

Cites

35 of 35 references verified0 citation errors · 0 not checkable

Statistics

Recomputing p-values needs the paper’s full text.

Claims the paper makes

We couldn’t retrieve this paper’s full text, so its claims and self-check weren’t run. The registry and citation checks still apply.

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The relevance of observations that purified endogenous molecules induce pro-inflammatory cytokines through TLR2 and/or TLR4 has been questioned because several of these proteins, including HSPs and HMGB1, bind lipopolysaccharide (LPS), making interpretation of pro-inflammatory responses difficult.

    “the relevance of these observations has previously been questioned because of the possibility of microbial contamination: several of these proteins, including HSPs and HMGb1, bind lipopolysaccharide (LPS), making the interpretation of pro-inflammatory responses by these molecules difficult28–30.”

    From Sterile inflammation: sensing and reacting to damage (2010) · cites this paper as [28]

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Gao, B., & Tsan, M.-F.
Affiliations
United States Department of Veterans Affairs (US)
Corresponding author
Gao, Baochong
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2003-01
Volume
278
Issue
1
Pages
174-179
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m208742200
OpenAlex
W2005570998
Cited by
396
References
35
Citations by year
2026: 3 · 2025: 3 · 2024: 3 · 2023: 5 · 2022: 3 · 2021: 5 · 2020: 5 · 2019: 10 · 2018: 11 · 2017: 11 · 2016: 12 · 2015: 10 · 2014: 19 · 2013: 16 · 2012: 21
FWCI
10.41
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
endotoxin contamination, lipopolysaccharide, TNF-α, macrophages, RAW 264.7 macrophages, CD14, TLR4, clathrin-coated vesicles, protein purification, endotoxin removal
MeSH terms
Animals, Anti-Bacterial Agents, Cell Line, Clathrin, Endotoxins, Hot Temperature, Humans, Lipopolysaccharides, Macrophages, Polymyxin B, Recombinant Proteins, Tumor Necrosis Factor-alpha, HSP70 Heat-Shock Proteins, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review