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CRITICAL CARE AND TRAUMA

Drotrecogin Alfa (Activated) Does Not Affect Intracellular Production of Interleukin-6 and Tumor Necrosis Factor- in Endotoxin-Stimulated Human Monocytes

Elke G. Czeslick, MD^*, Frank Nestler^*, Andreas Simm, PhD, Anja Struppert^*, and Armin Sablotzki, MD, PhD^*

Departments of *Anesthesiology and Critical Care Medicine and Cardiac and Thoracic Surgery, Martin Luther University Halle, Germany

Address correspondence and reprint requests to Elke Czeslick, MD, Department of Anesthesiology and Critical Care Medicine, Martin Luther University Halle, Ernst-Grube-Str. 40, 06120 Halle/Saale, Germany. Address e-mail to elke.czeslick{at}medizin.uni-halle.de.

	Abstract

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In this ex vivo laboratory study, we investigated the effects of drotrecogin alfa (activated) (DA(a)), a recombinant form of human activated protein C, on the intracellular expression of interleukin (IL)-6 and tumor necrosis factor (TNF)- on lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system. Whole blood samples from 10 healthy volunteers were stimulated with LPS (0.2 ng/mL) and incubated with 0.01, 0.1, 1, 10, and 100 nM of (final concentration) DA(a) for 3 h at 37°C and 5% CO₂. Intracellular expression of IL-6 and TNF- was assessed by flow cytometry. Our investigation showed that DA(a), at any of the concentrations tested, did not affect intracellular IL-6 and TNF- production in LPS-stimulated human monocytes after 3 h of incubation. The results of this investigation led us to conclude that any antiinflammatory activity of DA(a), if present, does not occur via detectible decreases in the production of proinflammatory cytokines IL-6 and TNF- in human monocytes.

	Introduction

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The Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) trial demonstrated the therapeutic efficacy in severe sepsis of a recombinant form of an endogenous anticoagulant, activated protein C (APC) (generic name, drotrecogin alfa [activated] [DA(a)]). Administration of DA(a) significantly reduced overall 28-day mortality compared with placebo (24.7% versus 30.8%) in patients with severe sepsis (n = 1690) (1). The evidence for a direct antiinflammatory effect of APC and the physiological significance of such an observation has been controversial. Data from Schmidt-Supprian et al. (2) indicated that the suggested antiinflammatory effect of APC is caused by a direct inhibition of the release of tumor necrosis factor (TNF) and macrophage migration inhibitory factor. Yuksel et al. (3) demonstrated that APC inhibited lipopolysaccharide (LPS)-induced TNF- production in human monocytes. In contrast, Derhaschnig et al. (4) showed that recombinant human APC did not alter systemic inflammation, as measured by interleukin (IL)-6 and TNF- release.

An evaluation of the pharmacodynamic effects of DA(a) on soluble inflammation biomarkers in the PROWESS trial (5) revealed, among other things, no significant differences in observed TNF- values between treatment groups. A significant reduction in IL-6 was detected only upon using the last-observation-carried-forward method of imputation in change from baseline and percent change from baseline analyses, suggesting that the antiinflammatory activity of DA(a) may not be detectible using a systemic biomarker approach.

There are no available data using flow cytometry to investigate the influence of DA(a) on human monocyte intracellular cytokine production at the single-cell level. Based on this, we performed the present investigation to evaluate the effect of DA(a) on the production of proinflammatory cytokines IL-6 and TNF- in endotoxin-stimulated human monocytes using fluorescence-activated cell sorter analysis (FACS).

	Methods

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The Ethics Committee of the Martin Luther University Halle approved this investigation. After informed consent, IV blood samples (200 µL) from 10 healthy volunteers (average age, 28 ± 2 yr), recruited from department personnel, were collected in sterile tubes (Sarstedt, Nuernbrecht, Germany) prefilled with Lepirudin (Refludan®; Schering AG, Berlin, Germany) (final concentration, 1 µg/mL). Blood samples were processed immediately under sterile conditions (Clean bench; Heraclean, Heraeus, Germany) according to a standard protocol. Blood was stimulated with LPS (055:B5) from Escherichia coli (SIGMA, Deisenhofen, Germany) (final concentration, 0.2 ng/mL) and incubated with DA(a) (Xigris®, Lilly, Indianapolis, IN) (final concentrations in each experiment were 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM). Samples were incubated at 37°C in a CO₂ (5%) humidified atmosphere for 3 h. After stimulation, the cell samples were prepared according to our standard procedure for FACS analysis (6,7).

Flow cytometric analyses were performed with a FACS-Calibur (Becton Dickinson, Heidelberg, Germany) and the Cellquest 3.3 software. The CD14⁺ monocytes were identified by immunofluorescence. At least 20,000 CD14⁺ monocytes were analyzed per sample. The CD14⁺ monocytes were gated for subsequent measuring of intracellular cytokines with phycoerythrin-labeled antibodies. Unstimulated samples and isotype controls were used as negative controls.

The number of measurements per experiment was 10. For analysis, the number of cytokine-positive monocytes and mean fluorescence intensities was assessed. Statistical analyses were performed with SPSS (Windows version 11.0.1; SPSS Inc., Chicago, IL). Normal distribution of data was examined with the Shapiro-Wilk test. Data values for cytokine-positive monocytes and total fluorescence values were taken as mean ± se of the mean (sem).

	Results

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DA(a) did not affect IL-6 or TNF- production (Figs. 1 and 2) in LPS-stimulated human monocytes, as indicated by the percent CD14⁺ cells (Table 1) and mean fluorescence intensity (Table 2) values obtained from identical experiments with varying concentrations of added DA(a). In all experiments, there was no effect of DA(a) (final concentration, 100 nM) on IL-6 or TNF- production in human monocytes that was not LPS-stimulated (data not shown). The percentage of monocytes (9.3% ± 1.5%), which was comparable with the percentage of monocytes in the white blood cell counts under clinical conditions, did not change during incubation with DA(a).

View larger version (33K): [in this window] [in a new window]   Figure 1. Expression of tumor necrosis factor (TNF)- in human monocytes after 3 h of stimulation with lipopolysaccharide (LPS) (0.2 ng/mL) as control value demonstrated in the fluorescent histogram of one representative experiment (original data).

View this table: [in this window] [in a new window]   Table 1. Interleukin (IL)-6 and tumor necrosis factor (TNF)- values from CD14+ cells after 3 h of stimulation with lipopolysaccharide (LPS) (0.2 ng/mL) and incubation with drotrecogin alfa (activated) (DrotAA)

View this table: [in this window] [in a new window]   Table 2. Interleukin (IL)-6 and tumor necrosis factor (TNF)- staining (mean fluorescence intensity) of CD14 monocytes after 3 h of stimulation with lipopolysaccharide (LPS) (0.2 ng/mL) and incubation with drotrecogin alfa (activated) (DrotAA)

View larger version (33K): [in this window] [in a new window]   Figure 2. Histogram of one representative experiment (original data) demonstrates no change in expression of tumor necrosis factor (TNF)- after stimulation with lipopolysaccharide (LPS) (0.2 ng/mL) and incubation with drotrecogin alfa (activated) (DA(a); 1 nM) for 3 h.

	Discussion

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Our investigation sought to analyze the influence of various DA(a) concentrations on the production of the proinflammatory cytokines IL-6 and TNF- in LPS-stimulated human monocytes. Our results, which for the first time used a flow cytometric technique in whole blood, demonstrate that DA(a) did not affect production of these important cytokines. Lepirudin (Refludan®) was used for anticoagulation to prevent any heparin-induced enhanced activity of the protein C inhibitor that could cause a markedly shortened half-life of DA(a).

In agreement with our results, Derhaschnig et al. (4) showed that DA(a) did not reduce LPS-induced inflammation as measured by TNF- and IL-6 release in 24 male volunteers. APC was shown to inhibit TNF- production in THP-1 cells stimulated with LPS (2). However, this effect was observed only when incubations were performed in serum-free media and not in the presence of 1%–10% serum. Serum-mediated inhibition could only be overcome by increasing APC concentrations far more than physiological levels (2). Instead of using monocytic cell lines, we used the ex vivo model of whole blood stimulated with LPS and incubated with the effector DA(a) immediately after drawing the blood sample. In contrast to isolated cells, whole blood retains all blood components, including serum, and maintains the in vivo ratios of the cells and noncellular components (8). This biological system permits interactions between the normal human blood constituents and mimics the physiological in vivo situation.

Schmidt-Supprian et al. (2) stimulated human monocytes with a concentration of LPS at 1 µg/mL; this was far more than the LPS concentration of 0.2 ng/mL we found to be effective for stimulation. A concentration of 0.2 ng/mL did not mask the influence of DA(a) by overstimulation of monocytes as a result of an excessive LPS concentration. 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 (7). Opal et al. (9) measured median endotoxin levels in patients with sepsis at 0.3 ng/mL. This LPS concentration was comparable to the concentration used for stimulation in our study.

The median blood levels of DA(a) in the PROWESS trial patients were approximately 45 ng/mL (55 ng/mL = 1 nM) (10). Concentrations of APC used with monocytes in most of the published in vitro studies (2,3,11) are quite large and considerably more than blood levels achieved with DA(a) in treated severe sepsis patients and in the ex vivo model we used for our evaluation.

Monocytes, a major source of proinflammatory cytokines, are an important part of the innate immune system. Using flow cytometry, we were able to demonstrate that physiological concentrations of DA(a) had no effect on production of IL-6 and TNF- in endotoxin-stimulated human monocytes. These results suggest that the proposed antiinflammatory effect of DA(a) is less important than its antithrombotic or fibrinolytic properties.

We would like to thank Dr. Paul Kretchmer (kretchmer{at}sfedit.net) at San Francisco Edit for his assistance in editing this manuscript.

	Footnotes

 
Accepted for publication June 14, 2005.

	References

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