© Birkhäuser Verlag, Basel, 2006 Inflamm. res. 55 (2006) 511–515 1023-3830/06/110511-05 DOI 10.1007/s00011-006-6057-3
Inflammation Research
E5564 (Eritoran) inhibits lipopolysaccharide-induced cytokine production in human blood monocytes E. Czeslick1, A. Struppert1, A. Simm2, A. Sablotzki1 1 Department of Anesthesiology and Critical Care Medicine, Martin Luther University Halle, Ernst-Grube-Str. 40, 06120 Halle/Saale, Germany, Fax:++049 345 557 2328, e-mail:
[email protected] 2 Department of Cardiac and Thoracic Surgery, Martin Luther University Halle, Germany Received 8 April 2006; returned for revision 13 June 2006; accepted by G. Wallace 7 July 2006
Abstract. Objective and design: In this ex vivo laboratory study, we investigated the effects of E5564 (eritoran), a tolllike receptor 4-directed endotoxin antagonist, on intracellular expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-a in lipopolysaccharide (LPS)-stimulated human monocytes assessed by flow cytometry. Material and method: Whole blood samples from 10 healthy volunteers (average age: 32 ± 2 years) were pre-incubated with 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 10 ng/ml E5564 for 45 min and after this stimulated with LPS (0.2 ng/ml), a dose we found to be the most effective for stimulation. Samples were incubated for 3 h at 37 °C and 5 % CO2. Intracellular expression of IL-6 and TNF-a was assessed by flow cytometry. Results: Our investigation showed that E5564 (0.03 ng/ml up to 10 ng/ml) caused a dose-dependent inhibitory effect on IL-6 and TNF-a production in LPS-stimulated human monocytes. Conclusions: The results of this investigation led us to conclude that E5564 has a remarkable LPS inhibitory activity manifested via down-regulation of the intracellular generation of pro-inflammatory cytokines IL-6 and TNF-a in human monocytes. Key words: E5564 – Proinflammatory cytokines – Monocytes – Flow cytometry
Introduction Bacterial lipopolysaccharide (LPS) (or endotoxin) is a major component of the outer cell membrane of Gram-negative bacteria with a remarkable potency as a signaling molecule that initiates the systemic inflammatory response by the innate immune system [1]. The concept of a very early removal Correspondence to: E. Czeslick
of this initiating agent to attenuate the excessive activation of innate immune responses accompanied by the release of pro-inflammatory mediators was viewed as a reasonable and optimal approach to sepsis but anti-endotoxin strategies to significantly reduce the mortality rate in human septic shock so far remain unproven [1–2]. E5564 (eritoran), a synthetic analog of the lipid A component of LPS, is a potent antagonist of LPS in several in vitro and in vivo models and is currently under clinical investigation as a possible therapeutic for the treatment of severe sepsis, septic shock and the inflammatory response to cardiopulmonary bypass surgery [3–5]. As an inhibitor of LPS-mediated stimulation of responsive cells in vitro, E5564 blocked the production of tumor necrosis factor (TNF)-a and other pro-inflammatory cytokines in human whole blood [6]. Monocytes as an essential part of the innate immune system are a major source of pro-inflammatory cytokines after stimulation with LPS [7–9]. There are no available data using flow cytometry to investigate the influence of E5564 on human monocyte intracellular cytokine production at the single-cell level. Based on this, the present investigation was performed to evaluate the hypothetical effect of E5564 (eritoran) on lipopolysaccharide (LPS)-induced intracellular production of interleukin (IL)-6 and tumor necrosis factor (TNF)-a in human monocytes using fluorescence-activated cell sorter analysis (FACS). Materials and methods The Ethics Committee of the Martin Luther University Halle approved this investigation. Intravenous blood samples (200 µl) from 10 healthy volunteers (average age: 32 ± 2 years), recruited from department personnel, were taken into sterile heparin-prefilled tubes (Sodium-Heparin NH/7.5 ml Monovette®, Sarstedt, Nuernbrecht, Germany) and processed immediately under sterile conditions (Clean bench, Heraclean, Heraeus, Germany) according to a standard protocol [10]. To find out the most effective LPS concentration for stimulation, a dose-effect curve for TNF-a was created: 200 µl of whole blood were diluted 1:5 with 800 µl Roswell Park Memorial Institute (RPMI) 1640 Cell
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Statistics Number of measurements per experiment was n = 10. For analysis, cytokine-positive monocytes as well as mean fluorescence intensities were assessed. Statistical analyses were performed with SPSS (Window version 11.0.1, SPSS Inc., Chicago, IL, USA). Normal distribution of data was examined with the Shapiro-Wilk test. Data values for cytokinepositive monocytes and total fluorescence values were taken as mean ± standard error of the mean (SEM). The Figures were created with Origin from OriginLab 7.5 (Northampton, MA, USA). The LPS concentration that stimulated 50 % of the monocytes to produce TNF-a (ED50) was calculated using nonlinear regression (MicrocalOrigin).
Results Figure 1 shows the LPS dose-effect curve for TNF-a. Its production decreased markedly when monocytes were stimulated with LPS concentrations between 1 and 0.1 ng/ml. The LPS concentration that stimulated 50 % of the monocytes to produce TNF-a (ED50) was calculated to be 0.19 ng/ml. Therefore, 0.2 ng/ml final concentration LPS was applied to stimulate human monocytes in further experimental settings. E5564 (0.03 to 10 ng/ml) inhibits IL-6 production in LPS stimulated human monocytes in a dose-dependent fashion (Fig. 2a) from 65.16 ± 5.53 % IL-6 positive cells (IL-6+) as control down to 43.81 ± 7.69 % IL-6+ when incubated with E5564 (0.03 ng/ml) and decreasing to 3.66 ± 0.12 % IL-6+ with E5564 (10 ng/ml). When assessing mean fluorescence intensities for IL-6 (Fig. 2b), the results were the same compared with data analysis of the percentage of cytokine-positive cells. Placebo (0.1, 1, 100 ng/ml final concentrations) did not influence IL-6 expression after LPS stimulation (Fig. 3). E5564 (0.03 to 10 ng/ml) was found to significantly inhibit TNF-a expression of human monocytes in a dosedependent manner (Figs. 4 and 5) from 68.38 ± 6.7 % TNF-
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Culture Medium (GIBCO BRL/Life Technologies, Karlsruhe, Germany) (with Glutamine) in 5 ml Falcon® polystyrene tubes (Becton Dickinson, Heidelberg, Germany). Thereafter 1 µl of GolgiPlugTM (PharMingen, Becton Dickinson, Heidelberg, Germany), a protein transport inhibitor containing Brefeldin A, was added to each tube. Blood samples were stimulated with LPS (final concentrations in each experiment were 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 ng/ml) (055:B5) from Escherichia coli (SIGMA, Deisenhofen, Germany) at 37 °C and CO2 (5 %) humidified atmosphere for 3 h. To investigate the influence of E5564 and Placebo on cytokine production, 200 µl of whole blood were incubated with E5564 (Eisai Research Institute, Andover, MA) (final concentrations in each experiment were 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 10 ng/ml) or Placebo (Eisai Research Institute) at 0.1, 1, 100 ng/ml (final concentration) 45 min before stimulation with LPS (055:B5) from Escherichia coli (SIGMA, Deisenhofen, Germany) (0.2 ng/ml final concentration) for each E5564 concentration. Samples were incubated at 37 °C and CO2 (5 %) humidified atmosphere for 3 h. Unstimulated samples were used to investigate the spontaneous cytokine release and for isotype controls. Furthermore the influence of E5564 (10 ng/ml final concentration) and Placebo (10 ng/ml final concentration) on cytokine production of unstimulated samples was assessed. After stimulation, the cell samples were prepared according to our standard procedure for fluorescence-activated cell sorter analysis (FACS): a) Cell surface staining: Cell samples were washed with 1 ml of CellWash (PharMingen, Becton Dickinson, Heidelberg, Germany) per tube once followed by centrifugation at 500 × g for 5 min. The supernatants were removed. The pellets were vortexed, washed with 1 ml of staining buffer (PharMingen, Becton Dickinson, Heidelberg, Germany) per tube, vortexed again, followed by centrifugation at 500 × g for 5 min. After removing the supernatants, a pellet of approximately 100 µl was vortexed again and 10 µl of Fluorescein isothiocyanate (FITC)-conjugated CD 14 (PharMingen, Becton Dickinson, Heidelberg, Germany) was added according to the protocol. The samples were stored at room temperature in the dark for 10 min. Thereafter the samples were washed with staining buffer twice. b) Intracellular staining: For fixation, the cell pellet was resuspended in 250 µl of Cytofix/Cytoperm (PharMingen, Becton Dickinson, Heidelberg, Germany) and stored at 4 °C in the dark for 10 min. Thoroughly resuspended fixed and permeabilized cells (100 µl) were mixed with 10 µl per tube Phycoerythrine (PE)-conjugated anti human IL-6 or TNF-a or appropriate isotype controls (PharMingen, Becton Dickinson, Heidelberg, Germany) and incubated at 4 °C in the dark for 30 min. After incubation, cells were washed two times with Perm/Wash-Buffer (PharMingen, Becton Dickinson, Heidelberg, Germany) and resuspended in 300 µl of staining buffer. c) Flow cytometry analysis: Flow cytometric measurement was performed with FACS-Calibur (Becton Dickinson, Heidelberg, Germany) and software Cellquest 3.3. CD14+ labelled monocytes were identified by immunfluorescence. At least 20,000 CD14+ monocytes were analyzed per sample. For measuring intracellular cytokines with PE labelled antibodies monocytes were gated from CD14+ cells. Unstimulated samples as well as isotype controls were used as negative controls.
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Fig. 1 Tumour necrosis factor (TNF)-a CD14+-positive cells after 3 h of stimulation with lipopolysaccharide (LPS) at different concentrations in whole blood samples. Values are mean ±standard error of the mean (SEM) of percentage positives in the CD14+ subpopulation.
Table 1. Interleukin (IL)-6 and tumor necrosis factor (TNF)-a values from unstimulated CD14+ cells after 3 h respectively 3 h of incubation with E5564 and Placebo. Data values were taken as mean ±standard error of the mean (SEM).
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0.40 ± 0.09 0.29 ± 0.07 0.40 ± 0.07
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Fig. 2. (a) Interleukin (IL)-6 CD14+-positive cells and (b) IL-6 staining (mean fluorescence intensity) after pre-incubation (45 min) of whole blood samples with E5564 (0.001 ng/ml, 0.003 ng/ml, 0.01 ng/ml, 0.03 ng/ml, 0.1 ng/ml, 0.3 ng/ml, 1 ng/ml and 10 ng/ml) followed by stimulation with lipopolysaccharide (LPS) (0.2 ng/ml) and 3 h of incubation. Values are (a) mean ±standard error of the mean (SEM) of percentage positives in the CD14+ subpopulation. **p < 0.01; ***p < 0.001 vs. control and (b) mean ±standard error of the mean (SEM). *p < 0.05; **p < 0.01 vs. control.
a positive cells (TNF-a+) as control down to 0.31 ± 0.07 % TNF-a+ when incubated with 10 ng/ml E5564. Mean fluorescence intensities for TNF-a followed the same significance (Fig. 4b). Placebo did not affect intracellular TNF-a production (data not shown). Table 1 demonstrates that there was no cytokine production without LPS stimulation. E5564 (10 ng/ml) as well as Placebo (10 ng/ml) revealed no effect on spontaneous cytokine release. Discussion Our investigation sought to analyze the influence of various E5564 concentrations on the production of the pro-inflammatory cytokines IL-6 and TNF-a in LPS-stimulated human
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Fig. 3. Histograms of one representative experiment (original data) demonstrate (a) Expression of Interleukin 6 (IL)-6 in human monocytes after 3 h of stimulation with lipopolysaccharide (LPS) (0.2 ng/ml) as control value (filled curve) and (b) no change in expression of IL-6 with Placebo (1 ng/ml) for pre-incubation (filled curve). (a)/(b) M1 represents the negative population with the isotype control (empty curve) and M2 fluorescence represents IL-6 positive cells.
monocytes. Our results, which for the first time used a flow cytometric technique in whole blood, demonstrate that nanomolar concentrations of E5564 blocked response to LPS at a dose of 0.2 ng/ml dose dependently. Wong et al. stimulated whole blood with a concentration of LPS at 1 and 10 ng/ml [4]; this was far more than the LPS concentration of 0.2 ng/ml we found to be effective for stimulation. We could show that LPS concentrations larger than 1 ng/ml did not result in a further increase of intracellular cytokine production in human monocytes. Opal and co workers measured median endotoxin levels in patients with sepsis at 0.3 ng/ml [11]. This LPS concentration was comparable to the concentration we used for stimulation in our study. In agreement with our results, Mullarkey et al. showed that E5564 10 nM (14 ng/ml) inhibited TNF-a production in human whole blood after LPS stimulation completely
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Fig. 4. (a) Tumour necrosis factor (TNF)-a CD14+-positive cells and b) TNF-a staining (mean fluorescence intensity) after pre-incubation of whole blood samples for 45 min with E5564 (0.001 ng/ml, 0.003 ng/ml, 0.01 ng/ml, 0.03 ng/ml, 0.1 ng/ml, 0.3 ng/ml, 1 ng/ml and 10 ng/ml) followed by stimulation with lipopolysaccharide (LPS) (0.2 ng/ml) and 3 h of incubation. Values are (a) mean ±standard error of the mean (SEM) of percentage positives in the CD14+ subpopulation. *p < 0.05; **p < 0.01; ***p < 0.001 vs. control and (b) mean ±standard error of the mean (SEM). *p < 0.05; **p < 0.01 vs. control. Counts
[6]. Compared to our investigation the LPS concentration of 10 ng/ml for stimulation was higher than in our experiments and cytokine levels were measured in plasma using Enzyme-linked Immunosorbent Assay (ELISA) method. By use of this method it is not possible to determine that specific cell population in whole blood, which is responsible for cytokine production. In contrast to isolated cells or cell lines, whole blood retains all blood components, including serum, and maintains the in vivo ratios of the cells and noncellular components [12]. This biological system permits interactions between the normal blood constituents and mimics the physiological in vivo situation. By use of the flow cytometric technique it is possible to reveal and analyze single cell level responses in the context of unselected cellular backgrounds. We therefore developed a system that employs stimulation of whole peripheral blood followed by identification of monocytes with monoclonal antibodies to their cell-surface mark-
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Fig. 5. Expression of tumor necrosis factor (TNF)-a in human monocytes after (a) 3 h of stimulation with lipopolysaccharide (LPS) (0.2 ng/ ml) as control value (filled curve) and (b) pre-incubation with 0.3 ng/ml (filled curve) respectively (c) 1 ng/ml E5564 followed by LPS stimulation (0.2 ng/ml) (filled curve). Fluorescent Histograms demonstrate one representative experiment (original data). (a)/(b)/(c) demonstrate overlays with isotype controls (empty curves) in M1 as negative population. M2 fluorescence represents the TNF-a producing positive population.
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ers and by detection of cytokines with intracellular staining as a physiological ex vivo approach [10]. The present investigation on the single cell level of human monocytes, performed with a different method of investigation, confirmed previous results that even low nanomolar concentrations of E5564 are capable of blocking response to LPS [4–6]. By use of flow cytometry we demonstrated that mean fluorescence intensity, representing the amount of cytokine produced per cell, as well as percent fluorescent to the percentage of monocytes that produce cytokine, were affected by E5564 in the same way. E5564 inhibits LPS action both in vivo and in vitro in whole blood assay systems [8]. Production of high levels of TNF-a and IL-6 was induced by LPS and was substantially inhibited by E5564. Despite inhibiting the production of these pro-inflammatory cytokines markedly, LPS-induced effects on vital signs and clinical symptoms were only moderately affected. Even low levels of pro-inflammatory cytokines are capable of causing extensive clinical effects (e. g. fever, tachycardia, leucopenia/leucocytosis) [8]. These symptoms may be more sensitive indicators of efficacy in blocking LPS [5, 8]. For that reason in order for E5564 to be effective in sepsis, applied doses should be sufficient to completely inhibit any activation of inflammatory response [5]. Rossignol et al. [3] determined 0.1 mg as the minimum effective dose of E5564 as the lowest dose that completely blocked all signs and symptoms of endotoxemia in healthy volunteers administered 4 ng/kg endotoxin. As a result of the reduced IL-6 and TNF-a release by >99 % the incidence of chills and headaches was decreased. Furthermore an absence of tachycardia and fever was observed. Rossignol and coworkers [3] concluded from the results of their preclinical and clinical studies that E5564 is a potent antagonist of LPS. The antagonism is dependent on the concentration of E5564 and LPS. The ex vivo analysis of the antagonistic activity of 0.5, 1.0, 2.0 and 3.5 mg E5564 over 30 min indicated that all doses completely or nearly completely blocked cytokine activation by 1 ng/ml LPS whereas less inhibition was noticed after challenge with 10 ng/ml of LPS. Mullarkey and co workers confirmed the toll-like receptor 4 as the target of LPS antagonism by E5564 [6]. Monocytes, a major source of pro-inflammatory cytokines, are an important part of the innate immune system. Using flow cytometry, we were able to demonstrate that
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E5564 is a highly active antagonist of LPS in an ex vivo whole blood setting by inhibiting intracellular IL-6 and TNF-a production of endotoxin-stimulated human monocytes in a dose-dependent manner. Acknowledgments. We gratefully acknowledge Dr. F. Gusovsky (PhD) from Eisai Research Institute, Andover, MA, USA for providing and sponsoring E5564 respectively Placebo and his very constructive discussion of our results.
References [1] Opal SM, Glück T. Endotoxin as a drug target. Crit Care Med 2003; 31 (1) (Suppl): S57. [2] Cross AS, Opal SM. Therapeutic intervention in sepsis with antibody to endotoxin: Is there a future? J Endotoxin Res 1994; 1: 57. [3] Rossignol DP, Lynn M. Antagonism of in vivo and ex vivo response to endotoxin by E5564, a novel synthetic lipid A antagonist. J Endotoxin Res 2002; 8 (6): 483. [4] Wong JN, Rossignol D, Rose JR et al. Safety, Pharmacokinetics, and Pharmacodynamics of E5564, a Lipid A Antagonist, during an Ascending Single-Dose Clinical Study. J Clin Pharmacol 2003; 43: 735. [5] Rossignol DP, Lynn M. TLR4 antagonists for endotoxemia and beyond. Curr Opin Investig Drugs 2005; 6 (5): 496. [6] Mullarkey M, Rose JR, Bristol J et al. Inhibition of Endotoxin Response by E5564, a Novel Toll-Like Receptor 4-Directed Endotoxin Antagonist. J Pharmacol and Exp Ther 2003; 304 (3): 1093. [7] Hagiwara E, Abbasi F, Mor G et al. Phenotype and frequency of cells secreting IL-2, Il-4, Il-6, Il-10, IFN and TNF-alpha in human peripheral blood. Cytokine 1995; 7 (8): 815. [8] Lynn M, Rossignol DP, Wheeler JL et al. Blocking of Responses to Endotoxin by E5564 in Healthy Volunteers with Experimental Endotoxemia. J Infect Dis 2003; 187: 631. [9] Lynn M, Wong N, Wheeler JL et al. Extended in Vivo Pharmacodynamic Activity of E5564 in Normal Volunteers with Experimental Endotoxemia. J Pharmacol and Exp Ther 2004; 308 (1): 175. [10] Czeslick EG, Simm A, Grond S, Silber RE, Sablotzki A. Inhibition of intracellular tumour necrosis factor (TNF)-a and interleukin (IL)-6 production in human monocytes by iloprost. Eur J Clin Invest 2003; 33: 1013. [11] Opal SM, Scannon PJ, Vincent JL et al. Relationship between Plasma Levels of Lipopolysaccharide (LPS) and LPS-Binding Protein in Patients with Severe Sepsis and Septic Shock. J Infect Dis 1999; 180: 1584. [12] West MA, Haegy W. Endotoxin tolerance: a review. Crit Care Med 2002; 30: 64.
