Elsevier

Immunology Letters

Volume 106, Issue 1, 15 July 2006, Pages 1-7
Immunology Letters

Current Views
“Dirty little secrets”—Endotoxin contamination of recombinant proteins

https://doi.org/10.1016/j.imlet.2006.04.007Get rights and content

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.

Introduction

Lipopolysaccharide (LPS), an integral component of the outer membrane of Gram negative bacteria, is a potent stimulant of the immune system [1]. LPS is the main cause of septic shock in humans, a syndrome characterised by the massive release of proinflammatory cytokines, activation of clotting and complement cascades, and activation of leukocytes. Following the discovery of Toll in Drosophila, the identification of a family of mammalian Toll-like receptors (TLRs) that recognise and signal in response to conserved molecular patterns (pathogen-associated molecular patterns) present within bacteria has transformed our understanding of innate immunity [2], [3], [4], [5], [6].

Section snippets

TLR4 and its agonists

Many TLR agonists are bacterial in origin reflecting the variety of microbial components that need to be recognised by the 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 downstream pathways including the NF-κB pathway, the MAP kinases, phosphatidylinositol-3′-kinase (PI-3K), and the interferon

The nature of the stimulus

Recently, a body of evidence has emerged to suggest that some of the effects previously attributed to the TLR4 agonists HSP60 and HSP70 may be the direct result of low amounts of endotoxin contained within the bacterial-derived recombinant proteins used experimentally to investigate their roles in vitro [34], [35], [36], though studies controlling carefully for endotoxin have still identified roles for these proteins in the activation of TLR4 [37]. There is also recent evidence that the heat

Detecting the contaminants

Given the potency of the many bacterial products that may contribute to signalling through one or more of the TLRs, it is vital that effects attributable to such products are differentiated from effects relating to the protein of interest. This relies on our ability not only to detect and quantify accurately the activity of the bacterial contaminants present but also to remove or neutralise them. It is important to emphasize that the methods available target LPS alone, thus failing to account

Decontaminating the contaminants

The removal of endotoxin from proteins is difficult [64]. If LPS is the only bacterial contaminant present, a number of methods have been described in the literature for its removal [65], [66], [67]. These include adsorption by activated carbon, anion-exchange chromatography, Triton X phase separation, the use of polymeric matrices and treatment with polymyxin B (PMB) or histamine-immobilised sepharose. Most have been designed with a clinical purpose in mind, producing products that do not

Future

The discovery of the TLRs is revolutionising our understanding of innate immunity and it is now clear that the TLRs represent a very specific mechanism for the recognition of bacterial molecular patterns. TLR signalling is complex but although much headway has been made, we still are far from understanding all the interactions that take place during signal transduction. TLR4 has been the centre of intense interest and a number of exogenous and endogenous agonists have been proposed. The full

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