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Published in final edited form as: Vet Immunol Immunopathol. 2007 Jan 31;116(3-4):182–9. doi: 10.1016/j.vetimm.2007.01.013

The cellular Toll-like receptor 4 antagonist E5531 can act as an agonist in horse whole blood

Clare E Bryant *,*, A Ouellette *, K Lohmann †, M Vandenplas †, J N Moore †, D J Maskell †, BA Farnfield †
PMCID: PMC7617840  EMSID: EMS206712  PMID: 17320193

Abstract

Sepsis and endotoxaemia are important causes of morbidity and mortality in humans. Research on sepsis focuses on rodent models most of which are poorly responsive to lipopolysaccharide (LPS), and thus do not mimic very well the high sensitivity of humans. Therefore there is a need to develop more clinically relevant models. Horses suffer from a similar endotoxaemic syndrome to humans with high morbidity and mortality. LPS analogues that act as antagonists at Toll-like receptor 4 (TLR4) are being developed as novel treatments for endotoxaemia. Due to differences in recognition of ligands by TLR4 from different mammalian species, individual LPS molecules may act as agonists in some species and antagonists in others. The synthetic lipid A analogue E5531 is an antagonist at TLR4 in humans and mice, but its effects at TLR4 from other species are unknown. In the studies reported here, Escherichia coli LPS is a full agonist on equine bone marrow macrophage-like cells and its effects are antagonised by E5531. Similarly, E. coli LPS is an agonist and E5531 an antagonist on monocytes isolated from peripheral blood of healthy horses and human embryonic kidney (HEK) cells, transiently transfected to express horse TLR4 and its associated cell surface proteins MD2 and CD14. In contrast, both E. coli LPS and E5531 behave as agonists in horse whole blood by inducing production of equivalent amounts of the inflammatory mediator prostaglandin. This finding suggests that modification of E5531 may occur in whole blood, for example deacylation, which alters its activity. This comparative study has revealed a novel pharmacological action of E5531 and emphasises the importance of extending studies of this nature beyond the normal rodent models.

Keywords: Toll-like receptor 4, E5531, horse, macrophage, whole blood assay, lipopolysaccharide

Introduction

Sepsis and endotoxaemia are important causes of morbidity and mortality in humans. Research in this area primarily has utilized rodent models of endotoxaemia, but these species are relatively insensitive to the effects of lipopolysaccharide (LPS), particularly when compared with the sensitivity of people (Olson et al., 1995). Like humans, horses are exquisitely sensitive to LPS, are particularly susceptible to endotoxaemia and sepsis, and exhibit similar clinical signs and pathology to septic human patients (Moore and Barton, 1998). These animals therefore provide a unique opportunity for the provision of research models and clinical situations to evaluate novel therapies.

In both humans and horses the systemic inflammatory response to bacteria and bacterial toxins results from the recognition of pathogen-associated molecular patterns (PAMPs) associated with the infectious agents by pattern recognition receptors (PRRs). An important family of PRRs is comprised by the Toll-like receptors (Beutler et al., 2003). Members of this family recognize bacterial PAMPs such as LPS (TLR4), bacterial lipoproteins and peptidoglycans (TLR2), bacterial flagellin (TLR5) and bacterial DNA (TLR9) (Beutler et al., 2003). Lipid A, which is the key toxic structure in LPS, initiates a systemic inflammatory response via a receptor protein complex comprising TLR4 and its associated proteins, CD14 and MD2, although other receptors may also recognize LPS (Beutler et al., 2003).

There are considerable differences between mammalian species in recognition of LPS from different bacterial species. For example, LPS from Rhodobacter sphaeroides (RSL) is an agonist in the horse and hamster, but an antagonist in humans and mice (Lien et al., 2000; Lohmann et al., 2003). In mice, ligand recognition of different LPS partial structures and analogues, such as Lipid IVa (lipid A with 4 acyl chains as opposed to the 6 acyl chains present in E. coli lipid A) and the yew tree derivative taxol, is dependent on the association of MD2 with TLR4 (Akashi et al., 2001). In addition to being dependent on MD2, humans recognize different modifications of LPS, such as changes in acylation, at a hyper-variable region of the TLR4 protein (Hajjar et al., 2002). Activation of TLR4 by E. coli lipid A is species-independent, at least in those species tested, but recognition of Salmonella enterica lipid A is species-specific, with the specificity being dependent on both TLR4 and MD2 (Muroi and Tanamoto, 2002). Little is known about the recognition of different bacterial lipid As by TLR4 in mammalian species other than humans, mice or hamsters, although it has been established that equine monocytes are activated by RSL (Lohmann et al., 2003).

E5531 is a synthetic compound based on the lipid A structure of Rhodobacter capsulatus, which shows much similarity in activity to RSL(Christ et al., 1995). R. capsulatus lipid A acts as an LPS antagonist in murine and human cells and whole blood and analogues of this compound currently are being investigated as treatments for sepsis in people (Bunnell et al., 2000; Kawata et al., 1999; Lien et al., 2000). To characterize E5531 in a species, other than the human, that is sensitive to LPS we have performed a range of in vitro assays using horse cells, transfected cells and horse blood. We have measured the effect of E5531 on the biosynthesis of prostaglandin (PG) in a modified whole blood assay (Jordan et al., 2000) and from an equine bone marrow macrophage-like (eCAS) cell line. We have also measured the effect of E5531 on the production of tumor necrosis factor (TNF)α by primary horse monocytes. In addition, using transient transfection techniques, we have reconstituted signaling through equine TLR4 in human embryonic kidney cells. The results of these studies show that E5531 inhibits the effects of E. coli LPS in the eCAS macrophage-like cell line, primary monocytes and transfected HEK cells, but is a potent agonist in horse whole blood assays. As Lipid IVa is a partial agonist in cellular models, it is possible that E5531 is deacylated in horse whole blood generating a partial structure similar to Lipid IVa. In conclusion we reveal an unexpected agonist effect of E5531 in whole horse blood. This indicates that careful research on the species-specific nature of the interactions of potential antagonists with TLR4 is necessary if the true range of pharmacological activities of particular ligand-receptor interactions is to be defined.

Materials and methods

Cell harvest, culture and materials

Horse monocytes were isolated from freshly collected whole blood (2.5 nmol EDTA per ml blood) by density gradient centrifugation and selective adherence to polystyrene as previously described (Henry and Moore, 1988). After density gradient centrifugation, 3x106 mononuclear cells were added to each well of a sterile 12-well polystyrene plate and incubated for 2 hours at 37ºC, 5%CO2 in air. Non-adherent cells were removed by washing 3 times with sterile phosphate buffered saline (PBS, Sigma Chemical Co., St. Louis, MO, USA). Monocytes were then incubated in RPMI-1640 (Bio Whittaker, Walkersville, MD, USA) with 5% fetal bovine serum (HyClone Laboratories Inc., Logan, UT, USA) and 1% penicillin/streptomycin solution (10,000 units/ml penicillin, 10 mg/ml streptomycin, Sigma Chemical Co., St. Louis, MO, USA). Cells were stimulated for 6 hours and cell media collected for TNFα assay.

Horse eCAS cells (Werners et al., 2004) were cultured at 37°C and 5% CO2 in RPMI (Gibco, UK) containing 20% horse serum, 1% penicillin (100U/ml) /streptomycin (100μg/ml), 1% L-glutamine (2mM), 1% non-essential amino acids (Gibco, UK), 1% sodium pyruvate (100mM) and 0.05 % amphotericin B (5mg/ml). RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum supplemented with 2mM glutamine, 200U/ml penicillin and 100μg/ml streptomycin. Human embryonic kidney (HEK293) cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum supplemented with 2mM glutamine, 200U/ml penicillin and 100μg/ml streptomycin.

In the studies with eCAS and RAW cells, 2x105 cells in 200µl of tissue culture medium were added to each well on a 96 well plate. When cells reached 90-100% confluency, they were incubated with either LPS (E. coli serotype 0127:B8; 0.01ng-1µg/ml) or E5531 (0.1-10µM). Cells were treated with LPS for 24 h and the medium collected to determine PG, TNFα or nitrite concentration. For studies with E5531, cells were pre-treated with E5531 or placebo (0.1-10 µM) for 1h prior to the addition of LPS. In the studies with IFN-γ, equine IFN-γ was added to the eCAS cells at a final concentration of 5IU/ml and murine IFN-γ was added to the RAW cells at 10IU/ml a maximum of 1h prior to ligand addition. HEK293 cells were plated out at 4x104 per well of a 96 well plate 24h prior to transfection. Cells were transfected with Polyfect (Qiagen, UK) according to the manufacturer’s instructions, using horse pcDNA3-TLR4 (1ng), pcDNA3-CD14 (1ng), pEFBOS-MD2 (1ng), pRL-TK (a constitutively active reporter construct that indicates cells have been transfected; 5ng; Promega) and pELAM (a nuclear factor kappa B reporter which when stimulated by LPS via TLR4 indicates pro-inflammatory gene expression; 50ng; Promega) per well. 48h after transfection, cells were stimulated with ligands for 4h. At the end of stimulation, cells were lysed with RLT buffer (Promega) and assayed for firefly luciferase and renilla luciferase activity using the Dual Luciferase assay (Promega). Firefly luciferase activity was divided by renilla luciferase activity to yield relative light units (RLU). Fold increase in RLU over baseline was calculated between stimulated samples and control samples exposed to culture medium alone.

All reagents were obtained from Sigma unless otherwise stated. E5531 was a gift from the Eiasi Coporation and horse IFN-γ was a gift from Dr F. Steinbach, Berlin. The human plasmids were generous gifts from Dr R. Medzhitov (TLR4), Dr P. Tobias (CD14) and Dr K. Miyake (MD2) respectively. The pBlueScript horse TLR4, CD14 and MD2 cDNA plasmids were generated by Dr M Vandenplas and subcloned into pcDNA3 (TLR4, CD14) or pFBOS (MD2).

Experimental animals

8 Welsh Mountain ponies of different sex and age, kept at grass, were used in the first part of study. 5 of these animals were used for the E5531 study. All procedures were performed under a Home Office licence in accordance with the Animal (Scientific Procedures) Act 1986.

Equine whole blood assay

Fresh blood was taken by venipuncture of the jugular vein and collected into tubes containing lithium heparin. Duplicate blood samples were diluted 1:1 in sterile heparinised (2 IU/ml) saline (0.9% sodium chloride). After addition of LPS (E. coli, serotype 0127:B8; 0-10 μg/ml), blood samples were incubated at 37°C for 24 hours. At the end of the incubation, the blood was centrifuged (10 min/13000rpm/20°C) and the plasma samples frozen for later analysis of prostaglandin (PG) concentrations by radio-immuno assay (RIA). To determine the effects of E5531 in equine whole blood, fresh blood samples were pre-incubated for one hour at 37 °C with E5531 (0.03-3 μM (0.05-5µg/ml) or placebo, 3µM), after which E. coli LPS (0-10 μg/ml) was added. The cells were then incubated for 24 hours at 37 °C. After centrifugation (10 min/13000rpm/20°C), the plasma was frozen for later analysis of PGs by RIA.

Radio-immuno assay for PG

Concentrations of the prostaglandins PGE2, PGI2 (assayed as its metabolite 6 keto PGF1α) and thromboxane (TX)B2 were determined by RIA. For each RIA a prostaglandin standard curve, total radioactivity and the non-specific binding of each [3H]-prostaglandin were determined. To each sample and standard (100 µl each), 200μl antiserum (anti-PG antisera; Sigma, UK) and 100μl [3H]-prostaglandin (0.0045 μCu/100μl; Amsersham-Pharmacia, UK) were added. The tubes were incubated for 18-24 hours at 0-4 °C. After incubation, 200 ml dextran-coated charcoal suspension (20 mg/ml charcoal, 4 mg/ml dextran) was added to all samples and controls, except totals and all tubes were centrifuged (3000 rpm, 4°C, 15 mins) to separate the bound and unbound fractions. The supernatant, containing the bound fraction, was decanted into scintillation-vials, 4 ml scintillant (Optiphase HiSafe II) was added and, after mixing, the radioactivity of each sample was determined by counting for one minute using a β-counter. Reference standards were prepared in radioimmunoassay buffer: phosphate buffered saline containing 0.1% bovine serum albumin and 0.1% sodium azide. To investigate the potential effect of the plasma matrix on antigen-antibody binding, the accuracy and precision of the assay was checked by conducting test assays on plasma spiked with known prostaglandin concentrations. These test assays were found to be both accurate and precise therefore in this particular radioimmunoassay the plasma matrix had no significant effect on antigen-antibody binding so plasma samples were not extracted.

TNFα bioassay

TNFα activity in equine samples was determined by an in vitro cytotoxicity bioassay as previously described (Morris et al., 1990).

Measurement of nitric oxide activity

To determine nitric oxide synthase (iNOS) activity, the supernatants from the cultured equine bone marrow cells or RAW 264.7 cells were removed 24h after addition of LPS and assayed for nitrite accumulation by the Griess reaction (Green et al., 1982). Briefly, an equal volume of Griess reagent (4% sulphanilamide and 0.2% naphtylethylenediamine dihydrochloride in 10% phosphoric acid) was added to an equal volume of sample and the colorimetric difference in optical density at 540 nm and 620 nm read immediately. The values obtained were compared to standards of sodium nitrite dissolved in DMEM and the concentration of nitrite released calculated and expressed as concentration (µM).

Data analysis

Data were analysed using ANOVA followed by Dunnett’s post test to compare all treated samples with a control. Significance was set at P < 0.05.

Results

E5531 inhibits the activation of eCAS cells and peripheral blood monocytes by E. coli LPS

Incubation of eCAS cells with E. coli LPS significantly increased production of both NO and PGI2 (Figure 1). E5531 was inactive when added to the cells in the absence of E. coli LPS, but antagonised the production of NO (Figure 2) and PGI2 (data not shown) in response to E. coli LPS. In parallel experiments, production of NO by the murine macrophage-like cell line RAW 264.7 (Royle et al., 2003) and production of TNFα by murine peripheral blood monocytes in response to E. coli LPS (Figure 2B) were also inhibited by E5531. Incubation of monocytes with E5531 alone had no effect on supernatant concentrations of TNFα.

Figure 1.

Figure 1

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A The effect of LPS on nitrite production, as a measure of NO release, from horse bone marrow derived cells. LPS (0-10μg/ml) was incubated with horse bone marrow cells for 24h at 37° C. After this time the cell supernatant was collected and the concentration of nitrite analysed by the Griess assay. Data represents the mean and standard deviation of at least 4 independent experiments. An * indicates that the nitrite production was significantly greater than the nitrite production from unstimulated cells at a level of p<0.05.

B The effect of LPS on PGI2 production from equine bone marrow-derived cells. LPS (0-10µg/ml) was added to equine bone marrow-derived cells and incubated for 24h at 37° C. After this time the cell supernatant was collected and the concentration of 6keto-F1α analysed by radio-immunoassay. Data represent the mean and standard deviation of at least 4 independent experiments. An * indicates that the 6keto-F1α production was significantly greater than the 6keto-F1α production from unstimulated cells at a level of p<0.05.

Figure 2.

Figure 2

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A The effect of E5531 on LPS-induced nitrite production, as a measure of NO release, from horse bone marrow derived cells 

The eCAS cells were pre-incubated for 1h with E5531 (0.03-3µM) and then treated with LPS (10µg/ml) for 24h at 37° C. After this time the cell supernatant was collected and the concentration of nitrite analysed by the Griess assay. Data represent the mean and standard deviation of at least 4 independent experiments.

B The effect of E5531 on LPS-induced TNFα production from primary horse monocytes 

The monocytes were pre-incubated for 1h with E5531 (0.03-3µM) and then treated with LPS (30ng/ml) for 4h at 37° C. After this time the cell supernatant was collected and the concentration of TNFα analysed by bioassay. Data represent the mean and standard deviation of at least 3 independent experiments.

Interferon-γ enhances the response of eCAS cells to E. coli LPS but has no effect on E5531

Low levels of ligand activity at TLR4 can be enhanced by priming cells with interferon (IFN)-γ (Bosisio et al., 2002). Pre-incubation of eCAS cells with IFN-γ (5IU) for 1h prior to the addition of LPS synergistically enhanced LPS-induced NO production (by 20±5%), but IFN-γ treatment of the cells did not result in the production of NO in response to E5531.

E5531 inhibits LPS-induced activation of the equine TLR4/MD2/CD14 complex

We used transient transfection assays to reconstitute LPS signaling in HEK293 cells through equine MD2, CD14 and TLR4 and measured activation of the ELAM promoter (an LPS sensitive gene). The ELAM promoter was activated by TNFα in HEK293 cells in the presence or absence of TLR4. E. coli LPS only activated the ELAM promoter in the presence of TLR4, CD14 and MD2, and E5531 inhibited this response (Figure 3). E5531 alone had no effect on the ELAM reporter in the presence or absence of equine TLR4, CD14 and MD2 (Figure 3).

Figure 3.

Figure 3

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The effect of LPS and E5531 on ELAM promoter activity in HEK cells transfected with horse TLR4, MD2 and CD14 

HEK cells were transiently transfected with ELAM firefly luciferase promoter construct, pRLTK renilla promoter construct, horse TLR4, MD2 and CD14. After 48h, cells were stimulated with LPS (10 ng/ml) or E5531 (10 µM) for 4h and luciferase and renilla activity assessed using a double luciferase assay (A). In a second set of experiments transfected cells were pre-incubated with E5531 (10 µM) for 1h then left unstimulated or stimulated with LPS for 4h (B). Data represent the mean and standard deviation of 3 (A) and 2 (B) independent experiments performed in triplicate.

E. coli LPS and E5531 induces the release of PGI2 and PGE2 but not TXA2 in equine whole blood

Nitric oxide measurement is unreliable in equine whole blood because of the variable nitrate content of grazing pasture (Farnfield et al., unpublished data) therefore we used PG detection as a measure of LPS-induced TLR4 activity. Untreated equine whole blood had low basal production of PGI2 (Figure 4A), PGE2 and TXA2 (data not shown), presumably through a COX-1-dependent mechanism. E. coli LPS (1 and 10µg/ml) caused a significant increase in production of PGE2 and PGI2 (p<0.05 and p<0.01 respectively; Figure 4A), but not TXA2 (data not shown). The increased production of PGI2 and PGE2 is likely to be due to COX-2 induction in the white blood cells, whereas production of TXA2 by platelets is through COX-1 only. In all further experiments using the equine whole blood assay, only the stable metabolite of PGI2 was measured. Treatment of equine whole blood with E5531 (0.3-3µM) significantly increased production of PGI2 (Figure 4B). When blood was pre-incubated with E5531 followed by E. coli LPS, the resulting release of the metabolite of PGI2 was greater than that caused by either ligand alone (data not shown).

Figure 4.

Figure 4

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A The effect of LPS on PG production in the equine whole blood assay. LPS (0-10μg/ml) was incubated with freshly collected equine whole blood for 24h at 37° C. After this time the plasma was collected and the concentration of 6keto-F1a analysed by radio-immune assay. Data represents the mean and standard deviation of 6keto-F1α after treatment with LPS from 8 horses (an * indicates that the 6keto-F1α production was significantly greater than the 6keto-F1α production from unstimulated blood at a level of p<0.05).

B The effect of E5531 on PG production in the equine whole blood assay. E5531 (0.03-3 µM) was incubated with freshly collected equine whole blood for 24h at 37 C. After this time the plasma was collected and the concentration of 6keto-F1a analysed by radio-immune assay. Data represent the mean and standard deviation of 6keto-F1α after treatment with LPS from 5 horses. An * indicates that the 6keto-F1α production was significantly greater than the 6keto-F1α production from unstimulated blood at a level of p<0.05.

Discussion

The results of the studies reported here reveal that E5531 is an agonist in horse whole blood. This is a novel pharmacological effect of E5531, a stable synthetic analogue of R. capsulatus lipid A that acts as a TLR4 antagonist in mice and humans. However, E5531 antagonises the effects of E. coli LPS in horse mononuclear phagocytes and in HEK293 cells transiently transfected with horse TLR4/MD2. The antagonist effects of E5531 are similar to results seen in vivo in mice, as well as in human whole blood and cells (Bunnell et al., 2000; Chow et al., 1999; Christ et al., 1995; Kawata et al., 1999). In equine whole blood the agonist effect of E5531 was comparable to that seen with E. coli LPS. Because E5531 is an LPS antagonist in human whole blood, the agonist effect of E5531 in horse blood appears to be species-specific rather than a preparation-specific phenomenon. These data are important because they show that biological activities of compounds acting at TLR4 cannot be extrapolated across species.

The mechanism underlying the agonist effect of E5531 in whole blood as opposed to its antagonist effect in isolated cell preparations is unclear. One possible explanation is that cytokines produced in whole blood upon stimulation with E5531 may alter cellular responsiveness to ligands such as LPS. Whole blood contains cells of the immune system that are capable of releasing a range of cytokines, such as IFN-γ, when activated by LPS. During sepsis or endotoxaemia, blood concentrations of IFN-γ are frequently increased. IFN-γ synergises with LPS to enhance macrophage activation (Adams and Hamilton, 1984; Nathan et al., 1984), and while there are no data in the literature to suggest that IFN-γ can cause E5531 to act as an agonist, co-administration of IFN-γ with the non-toxic LPS from R. capsulatus caused enhanced NO production by murine macrophages (Denlinger et al., 1998). IFN-γ increases both TLR4 protein production and its expression on the cell surface (Bosisio et al., 2002), and this up-regulation of TLR4 could presumably allow a low level of agonist activity to be revealed. In our experiments, however, E5531 remained an antagonist when eCAS cells were primed with IFN-γ however this does not preclude the possibility of the effects of other cytokines or plasma soluble factors that could explain our observations.

Another possible explanation for our results could be structural modification of E5531 in whole blood preparations. Whole blood contains neutrophils which will degranulate when stimulated with LPS to release a range of enzymes which could alter the biological activity of E5531 by several mechanisms such as oxidation or chlorination. E5531 is a synthetic analogue of R. capsulatus lipid A and contains six acyl chains (Christ et al., 1995). Lipid A compounds with reduced numbers of acyl chains have varying biological effects; for example, lipid IVa carrying only 4 acyl chains is an agonist in murine cells, an antagonist in human cells, but a partial agonist in horse cells (unpublished data) (Akashi et al., 2001; Hajjar et al., 2002; Lien et al., 2000). It is possible that horse blood contains a lipid A deacylase enzyme (Munford and Hunter, 1992), that alters E5531 to a Lipid IVa-like structure having agonist activity. As yet, there is no evidence to support this hypothesis and further experiments investigating the effect of the different cell types or enzymes present in whole blood on E5531 would be a useful follow-up study to this study.

In conclusion, we show that E5531, despite being an antagonist in horse cellular models, has the novel pharmacological effect of being an agonist in horse whole blood. This comparative study has revealed a novel pharmacological action of E5531 and emphasises the importance of this comparative approach to biological studies.

Acknowledgements

This work was supported by a Wellcome Trust Advanced Fellowship award to CEB. We would like to thank the Eisai Corporation and Dr F. Steinbach for the gift of E5531 and equine IFN-γ, respectively. We further thank Professor Christian Raetz for discussions on this manuscript.

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