Immunology Letters 106 (2006) 1–7 Current Views “Dirty little secrets”—Endotoxin contamination of recombinant proteins Sonia J. Wakelin a,d,∗ , Ian Sabroe c , Christopher D. Gregory a , Ian R. Poxton b , John L.R. Forsythe d , O. James Garden d , Sarah E.M. Howie a,e a MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh EH8 9AG, UK b Medical Microbiology, Medical School, University of Edinburgh, Edinburgh EH8 9AG, UK c Division of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK d School of Clinical Sciences and Community Health, Clinical and Surgical Sciences (Surgery), Royal Infirmary, 51 Little France Crescent, Edinburgh EH16 4SA, UK e Division of Pathology, School of Molecular and Clinical Medicine, University of Edinburgh, Edinburgh EH8 9AG, UK Received 29 December 2005; received in revised form 19 April 2006; accepted 21 April 2006 Available online 11 May 2006 Abstract The identification of Toll-like receptors has revolutionised our understanding of innate immunity. TLR4 transduces the LPS signal and that of a number of structurally and functionally unrelated agonists. However, recent evidence adds to longstanding concerns that endotoxin contamination of bacterially derived recombinant TLR4 agonists is responsible for effects attributed to these molecules. We highlight key factors in differentiating specific agonist effects from those of endotoxin and emphasize why conventional methods of detecting and eliminating LPS may lead to erroneous results. We propose that considerable caution is needed in the investigation of TLR4 agonists, particularly when using proteins produced in a bacterium that also houses the most ideal TLR4 agonist, LPS. © 2006 Elsevier B.V. All rights reserved. Keywords: Toll-like receptors; LPS; Endotoxin; Recombinant proteins 1. Introduction Lipopolysaccharide (LPS), an integral component of the outer membrane of Gram negative bacteria, is a potent stim- ulant of the immune system [1]. LPS is the main cause of septic shock in humans, a syndrome characterised by the mas- sive release of proinflammatory cytokines, activation of clotting and complement cascades, and activation of leukocytes. Fol- lowing the discovery of Toll in Drosophila, the identification of a family of mammalian Toll-like receptors (TLRs) that recog- nise and signal in response to conserved molecular patterns (pathogen-associated molecular patterns) present within bacte- ria has transformed our understanding of innate immunity [2–6]. 2. TLR4 and its agonists Many TLR agonists are bacterial in origin reflecting the vari- ety of microbial components that need to be recognised by the ∗ Corresponding author. E-mail address: Sonia.Wakelin@btinternet.com (S.J. Wakelin). innate immune system (Fig. 1). Upon activation, TLRs signal as dimers, complexing via their intracellular Toll/interleukin- 1 receptor (TIR) domains with a family of adaptor proteins in TLR-specific patterns. This results in the activation of down- stream pathways including the NF-B pathway, the MAP kinases, phosphatidylinositol-3 ′ -kinase (PI-3K), and the inter- feron regulatory factors (IRFs). Pathway activation results in proinflammatory gene transcription and cell-type and TLR spe- cific activation phenotypes [4,7], enhancing antigen presenting activity and providing a key role for TLRs in linking innate and acquired immune responses [8]. Considerable attention has focused on TLR4 following exper- iments in the lipopolysaccharide (LPS)-hyporesponsive mouse strains C3H/HeJ and C57BL/10ScCr highlighting its impor- tance in LPS signal signalling [9,10]. TLR4 requires accessory molecules for optimal LPS signal transduction. Lipopolysaccha- ride binding protein (LBP) is an acute phase serum protein that facilitates transfer of LPS from its typical micellar structures into the cell membrane, where via CD14 it is presented to a signalling complex formed of MD-2 and TLR4 [11–14]. Other proteins such as CD11b/CD18 may also serve as coreceptors 0165-2478/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2006.04.007