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This article is part of the supplement: 33rd International Symposium on Intensive Care and Emergency Medicine

Poster presentation

Exploring the translational disconnect between the murine and human inflammatory response: in vitro analysis of the dose-response relationship of LPS and NFκB activation in murine and human immune cells

EP McCarron*, I Welters, D Williams, D Antoine and A Kipar

  • * Corresponding author: EP McCarron

Author Affiliations

University of Liverpool, UK

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Critical Care 2013, 17(Suppl 2):P18  doi:10.1186/cc11956

The electronic version of this article is the complete one and can be found online at: http://ccforum.com/content/17/S2/P18


Published:19 March 2013

© 2013 McCarron et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Inflammation, as seen in sepsis and systemic inflammatory response, is dependent on the activation of the NFκB pathway through Toll-like receptors (TLRs) [1]. Recreating an inflammatory response using lipopolysaccharide (LPS) can provide results that are different to clinical sepsis [2]. By examining NFκB activation in murine and human cells, a species comparison can be made to investigate differences at the cell level that may contribute to the translational disconnect seen in vivo.

Methods

THP1 human monocytes (passages 9 to 11) and RAW 264.7 murine macrophages (passages 15 to 20) were cultured in RPMI-1640 and DMEM respectively and then challenged with LPS. After settling for 24 hours, cells were dosed with six or seven doses of LPS. After 1 hour, nuclear extraction and proteins were separated by acrylamide gel electrophoresis. Membranes where then immunoblotted for actin and p65, followed by densitometric analysis in order to quantify the amount of p65 that had translocated from the cytoplasm to the nucleus (by subtraction from consistent nuclear actin).

Results

Murine cells required higher doses of LPS compared with human cells in order to detect p65 (human, 1 pg/ml to 100 ng/ml; murine, 30 pg/ml to 1,000 ng/ml). THP1 cells showed a greater fold increase in the p65:actin ratio compared with RAW 264.7 cells. Human cells responded to lower concentrations of LPS. Murine cells appeared to show a molecular resistance to lower doses, but their response was very sensitive at higher doses. A dose-response relationship of LPS dosing and NFκB activation was observed in both cell lines.

Conclusion

Immunoblotting for p65 is a reliable and reproducible method to determine NFκB activation in cultured cells. Macrophages are more responsive to LPS than monocytes [3] so differences between cell lines would have been expected to be the reverse of what was observed. The species difference in response to LPS may contribute to the apparent disconnect between human and murine responses to LPS and may partially explain the difficulties of translating therapeutic interventions into clinical human sepsis.

References

  1. Bonizzi G, Karin M:

    Trends Immunol. 2004, 25:280-288. PubMed Abstract | Publisher Full Text OpenURL

  2. Remick DG, Ward PA:

    Shock. 2005, 24(Suppl 2):7-11. PubMed Abstract | Publisher Full Text OpenURL

  3. Takashiba S, et al.:

    Infect Immun. 1999, 67(11):5573-5578. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL