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Originally published online as doi:10.1189/jlb.0707497 on February 27, 2008

Published online before print February 27, 2008
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(Journal of Leukocyte Biology. 2008;83:1388-1395.)
© 2008 by Society for Leukocyte Biology

Selenium supplementation induces metalloproteinase-dependent L-selectin shedding from monocytes

Ingo Ahrens*,{dagger},1, Christoph Ellwanger*, Belinda K. Smith{dagger}, Nicole Bassler{dagger}, Yung Chih Chen{dagger}, Irene Neudorfer*, Andreas Ludwig{ddagger}, Christoph Bode* and Karlheinz Peter{dagger}

* Department of Cardiology and Angiology, University Hospital of Freiburg, Freiburg, Germany;
{dagger} Department of Atherothrombosis and Vascular Biology, Baker Heart Research Institute, Melbourne, Victoria, Australia; and
{ddagger} Institute for Molecular Cardiovascular Research (IMCAR), University Hospital Aachen, Aachen, Germany

1Correspondence: Centre for Thrombosis and Myocardial Infarction, Baker Heart Research Institute, 75 Commercial Road, Melbourne VIC 3004, Australia. E-mail: ingo.ahrens{at}baker.edu.au


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ABSTRACT
 
Selenium therapy in patients with severe sepsis improves clinical outcome and has been associated with increased activity of the selenoprotein glutathione peroxidase. However, the mechanism of the observed beneficial effects remains unclear. We determined the effect of selenium treatment on the monocyte adhesion molecule L-selectin and L-selectin-related monocyte functions in vitro and transferred our findings to an in vivo mouse model. Monocytes were purified, cultured, and incubated in the presence or absence of supplemented selenium and metalloproteinase (MP) inhibitors for up to 16 h. Expression of L-selectin was unaffected after 2 and 6 h but decreased after 16 h of incubation in the presence of selenium. Soluble L-selectin (sL-selectin) in the supernatant was determined by ELISA. A 2.3-fold increase as a result of shedding of L-selectin was observed after 16 h of selenium treatment. Addition of the MP inhibitors GM6001, TNF-{alpha}-converting enzyme inhibitor 2, or GW280264X strongly reduced selenium-induced L-selectin shedding, indicating a MP-dependent mechanism. The functional consequences of L-selectin shedding were examined in a flow chamber model. Selenium-treated monocytes showed significantly decreased rolling and adhesion to the L-selectin ligand Sialyl-Lewisa under conditions of venous shear stress (0.5 dyne/cm2). Selenium treatment of C57BL6 mice led to increased serum levels of sL-selectin, underscoring the in vivo relevance of our findings. We describe a selenium-induced down-regulation of L-selectin on monocytes as a consequence of MP-dependent shedding of this membrane-anchored adhesion molecule. The impairment of monocyte adhesion by selenium supplementation may represent an important, underlying mechanism for the modulation of inflammatory reactions in patients with severe sepsis.

Key Words: sepsis • sL-selectin • MMP


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INTRODUCTION
 
In humans, the biological actions of the trace element selenium are predominantly mediated by the incorporation of selenium into selenoproteins, a group of proteins comprising at least 30 known members [1 ]. The glutathione peroxidases (GPXs) represent one of the major families of selenoproteins and play a pivotal role in the antioxidant system of the cell. Decreased serum selenium levels have been observed and correlated with worse clinical outcomes in critically ill patients admitted to an intensive care unit [2 ]. Decreased selenium levels have also been noted in patients with severe inflammatory response syndrome (SIRS) [3 ]. Supplementation of selenium in a small pilot study of 42 patients with SIRS led to an increase in activity of the GPXs and to improved clinical outcomes [4 ]. Additionally, in a larger, randomized, multicenter trial, selenium treatment reduced mortality among patients with severe sepsis or septic shock [5 ]. SIRS is associated with an increase in the production of reactive oxygen species (ROS) [6 ], particularly by activated macrophages, contributing to host tissue injury through oxidative damage. It is this oxidative damage that is reduced by the enzymatic, antioxidant actions of GPX.

Tissue resident macrophages are derived from monocytes circulating in blood. Once monocytes adhere to endothelial cells and migrate toward tissues, they differentiate into macrophages. During infection, macrophages are the main effectors of innate immunity as a result of their capacity to ingest microbes, their antimicrobial activity, and their role as a major source of inflammatory mediators. L-selectin is a member of the selectin family and is expressed on leukocytes. The interaction of L-selectin with its endothelial cell ligands leads to leukocyte deceleration, tethering, and rolling along the capillary wall. Finally, firm adhesion of these cells to the endothelium is mainly mediated by the interaction of the β2-integrins on the leukocyte with endothelial cell adhesion molecules of the IgG family (ICAM) [7 , 8 ]. The ligands for the selectin family consisting of E-, L-, and P-selectin are predominantly mucin-like sialoglycoproteins such as P-selectin glycoprotein ligand 1 (PSGL-1). All selectins recognize the tetrasaccharide sialyl Lewis X, which is the binding determinant of the sialoglycoprotein ligands [9 ]. In lymph nodes, L-selectin recognizes ligands such as CD34 [10 ], podocalyxin, and endoglycan and mediates lymphocyte trafficking through these organs [11 ]. In the microvasculature, L-selectin facilitates neutrophil rolling and migration during inflammatory responses mediated through ligands expressed on leukocytes and endothelial cells such as PSGL-1 [12 ], mucosal addressin cell adhesion molecule-1 [11 , 13 ], and endothelial heparan sulfate [14 ].

L-selectin surface expression can be rapidly down-regulated by ectodomain shedding. TNF-{alpha}-converting enzyme [TACE; or a disintegrin and metalloprotease-17 (ADAM17)], which is a member of the disintegrin and metalloproteinase (MP; ADAM) family of MPs, is one of the major sheddases of L-selectin, cleaving the L-selectin receptor close to the cell membrane and generating a 65- to 95-kD soluble L-selectin (sL-selectin) [15 16 17 18 ]. Released sL-selectin purified from human serum was found to inhibit the attachment of lymphocytes to activated endothelial cells. This indicates that sL-selectin is biologically active and that high levels of sL-selectin may prevent overwhelming leukocyte recruitment to sites of inflamed endothelium [19 , 20 ]. However, there remains uncertainty about the full biological role of sL-selectin, although its importance is highlighted by studies showing that decreased, early serum sL-selectin levels lead to increased mortality in patients with SIRS [21 ]. Also, a recent meta-analysis suggested that early, decreased sL-selectin after multiple trauma predicts the occurrence of acute lung injury and acute respiratory distress syndrome [22 ].

The aim of our study was to investigate whether the observed, improved clinical outcome in patients with severe sepsis supplemented with selenium [4 ] relates to an effect on L-selectin expression and shedding from monocytes. We studied the relationship among L-selectin expression, sL-selectin generation, and functional consequences of selenium supplementation in isolated monocytes derived from healthy donors. We were able to demonstrate selenium-induced shedding of L-selectin from monocytes, leading to reduced rolling and adhesion of monocytes in vitro. Furthermore, we were able to detect a selenium-induced increase in sL-selectin in vivo in a mouse model. Thus, shedding of L-selectin may represent the underlying mechanism of selenium’s therapeutic effect on the inflammatory response in patients with severe sepsis.


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MATERIALS AND METHODS
 
Antibodies and reagents
FITC-conjugated anti-CD62 ligand (CD62L) mAb (FMC46) for flow cytometry was purchased from Dako Cytomation (Denmark). H-149 (mouse anti-human sL-selectin mAb) for immunoblotting was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and HRP-conjugated goat anti-mouse IgG antibody was obtained from Roche (Nutley, NJ, USA). Sodium selenit was purchased from Biosyn (Carlsbad, CA, USA) as a ready-made solution of 50 µg/ml in 0.9% NaCl. Matrix MP (MMP) inhibitors GM6001 and TACE inhibitor 2 (TAPI-2) were purchased from Calbiochem (San Diego, CA, USA). The hydroxamate MP inhibitors GI254023X and GW280264X were synthesized as described earlier [23 , 24 ]. TACE Substrate II was obtained from Calbiochem. Ficoll for the purification of monocytes was purchased from Biochrom (Cambridge, UK). RPMI-1640 medium and PBS were obtained from Cambrex (East Rutherford, NJ, USA). The instant ELISAs for the detection of sL-selectin were from Bender Medsystems (Burlingame, CA, USA). Sialyl Lewisa-poly[N-(2-hydroxyethyl)acrylamide]-biotin was used as a substrate for the coating of the parallel plate flow chamber and was obtained from Glycotech (Gaithersburg, MD, USA).

Isolation and cultivation of monocytes
Monocytes from healthy donors were isolated using Ficoll density gradient centrifugation (800 g, 20 min, 23°C) and transferred to a 75-ml tissue-culture flask. After incubation for 2 h in RPMI medium supplemented with 10% heat-inactivated FCS, 1% penicillin/streptomycin, and 1% L-glutamine, the supernatant was discarded, and the adherent monocytes were washed with PBS containing Ca2+ and Mg2+. For the experiments, the adherent monocytes were maintained in RPMI medium as described above at 37°C and 5% CO2 in a humidified atmosphere for 16 h, with or without supplementation of sodium selenit in the indicated concentrations (Biosyn).

Monocyte viability assay
Monocytes were purified by Ficoll gradient centrifugation and cultured in RPMI medium containing 10% FCS. Selenium concentrations from 1 to 12.5 µg/ml and incubation periods up to 16 h were assessed for cytotoxicity by MTT assay as described by Mosmann [25 ]. MTT (Sigma Chemical Co., St. Louis, MO, USA) is a pale, yellow, water-soluble substrate that is converted into a dark-blue, insoluble formazan. This conversion requires active mitochondria in living cells. After the incubation period, the supernatant was carefully removed, and the formazan crystals were dissolved in isopropanol/0.04 M hydrochloric acid (the latter preventing interference in the colorimetric reading by turning the color of the pH indicator phenol red in the RPMI culture medium into a yellow color that does not interfere with the wavelength used to read the blue color caused by the formazan). The absorbance was analyzed at 570 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

Detection of sL-selectin by ELISA
Monocytes were cultured in six-well plates (Costar, Corning, NY, USA) for 16 h at 37°C, with or without supplementation of selenium (2 µg/ml) and with or without GM6001 (100 µM), TAPI-2 (100 µM; a more specific inhibitor of ADAM17/TACE), GI254023X (10 µM), or GW280264X (10 µM) before transferring 500 µl each supernatant to Eppendorf tubes, which were centrifuged at 220 g for 5 min to obtain a cell-free supernatant. The supernatant (50 µl) was added to the 100-µl aqua dest.-containing wells of a 96-well plate of the sL-selectin instant ELISA (Bender Medsystems) following the manufacturer’s instructions. Light emission was measured at 450 nm using a microplate reader (Molecular Devices).

Detection of sL-selectin by Western blot
For Western blot analysis, 12 µl cell-free supernatant from cultured monocytes and 3 µl loading dye (containing 250 mM Tris, 50% glycerol, 10% SDS, 0.5% bromophenol blue, and 500 mM DTT) were mixed and incubated at 97°C for 5 min before being analyzed by 12% SDS gel electrophoresis. Separated proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA, USA), which was blocked in PBS–Tween–1% BSA for 1 h, followed by incubation with the mouse anti-human sL-selectin antibody (H-149, Santa Cruz Biotechnology; 1:1000) for another hour. Finally, the membrane was incubated with a goat anti-mouse HRP mAb (Roche; 1:5000) for 1 h. The membrane was finally washed twice with PBS–Tween (0.2%) for 10 min and twice with aqua dest. for 5 min. The chemiluminescence detection system (SuperSignal, Pierce, Rockford, IL, USA) was used to visualize the sL-selectin protein.

Detection of L-selectin and ADAM17/TACE expression by flow cytometry
Monocytes were cultivated for 16 h at 37°C and 5% CO2 in a humidified atmosphere, with or without supplementation of selenium (2 µg/ml, Biosyn). Thereafter, cultivated monocytes were washed and resuspended at 5 x 106/ml in PBS containing Ca2+ and Mg2+. The cell suspension (50 µl) was transferred to a FACS tube and incubated with 10 µl FITC-conjugated, anti-CD62L mAb (Clone FMC46, Dako Cytomation) for 15 min at room temperature or 10 µl FITC-conjugated anti-TACE mAb (Clone 111633, R&D Systems, Minneapolis, MN, USA) for 30 min at 4°C. PBS (200 µl) containing Ca2+ and Mg2+ was added to each sample before being analyzed by FACSCalibur (Becton Dickinson, San Jose, CA, USA).

ADAM17/TACE activity assay
Isolated monocytes (50 µl; 5x106/ml) were transferred to a 96-well plate and covered with 100 µl RPMI medium containing 10% FCS. After an adherence time of 2 h, the medium was removed, and the wells were washed with 100 µl PBS containing Ca2+ and Mg2+ and again covered with 100 µl RPMI containing 10% FCS. Following this, 100 µM TACE Substrate II (EMD, Waltham, MA, USA) was added, followed by a 16-h incubation at 37°C and 5% CO2 in a humidified atmosphere, with or without selenium (2 µg/ml, Biosyn). After 16 h, the supernatant was transferred to a fresh, 96-well plate before being quantified by a fluorescence-ELISA reader (Molecular Devices) with extinction at 320 nm and emission at 395 nm.

Flow chamber assay
Monocyte rolling and adhesion to a plastic surface coated with Sialyl-Lewisa (25 µg/ml, Glycotech) under conditions of low shear stress were investigated using a parallel plate flow chamber (Glycotech). Monocytes (80,000/ml) were perfused in PBS containing Ca2+ and Mg2+ with a low shear rate (0.5 dynes/cm2; corresponding to venous shear stress) for 5 min, followed by a period of 20 s at a higher shear rate (15 dynes/cm2; corresponding to arterial shear stress). Monocyte interaction with the coated surface was visualized by video-microscopy (AVT BC-11, AVT Horn, Germany) connected to an inverted microscope (Zeiss Axiovert 25, Zeiss, Thornwood, NY, USA) using a 10x objective magnification (Achromat). The number of rolling monocytes was determined as the mean value from video frames of 5 s duration at four different time-points, each of them 1 min apart. Adherent monocytes were counted at six different time-points (30 s, 1:30, 2:30, 3:30, 4:30, and 5:30 min), as described before [26 ].

In vivo sL-selectin shedding (mouse serum sL-selectin detection)
The Animal Care Committee of the University of Freiburg (Germany) approved the experimental protocol. Eight- to 9-week-old C57BL6 mice were anesthetized with ketamine (50 mg/Kg)/xylazine (10 mg/Kg) and then injected with sodium selenit (2 µg/ml estimated total blood volume) or equal amounts of saline via a catheter (26 gauge, BD Biosciences, San Jose, CA, USA) inserted into the tail vein. The bolus injection was then followed by a continuous infusion of 2 µg over a period of 60 min via a KdScientific 100 syringe pump. Animals were killed, and blood was taken by direct heart puncture at the end of the continuous infusion. Blood was left to coagulate for 2 h at room temperature, and thereafter, serum was separated by centrifugation for 20 min at 2000 g. Mouse sL-selectin was detected from the serum samples with an ELISA (Quantikine®, R&D Systems), following the manufacturer’s instructions.


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RESULTS
 
Effect of selenium on monocyte viability and activation status
To determine whether potential peak concentrations of selenium obtained during the bolus and infusion periods in the clinical trials are toxic to isolated monocytes, we applied a monocyte viability assay, based on the conversion of MTT to a dark-blue, insoluble formazan by mitochondria of living cells. Monocytes were incubated with increasing concentrations of selenium. Cytotoxicity was observed at concentrations above 6.25 µg/ml selenium (Fig. 1A ). An incubation period of 16 h at a concentration of 2 µg/ml, the concentration typically used in our experiments, did not result in monocyte toxicity.


Figure 1
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  		Figure 1. Selenium supplementation causes L-selectin shedding from monocytes, which were incubated with selenium at concentrations from 1 to 25 µg/ml for up to 16 h (A). Monocyte viability was evaluated by a MTT assay. A control culture of nontreated monocytes served as the 100% value. Every single value depicted was calculated as the percentage of living cells compared with the control culture (values are depicted as mean±SD; n=3). (B) Representative flow cytometry histograms for L-selectin expression after 2, 6, and 16 h of incubation in the absence (1) and presence (2) of selenium (2 µg/ml). Down-regulation of L-selectin occurred after 16 h of treatment. (C) Increased sL-selectin was determined by ELISA in the supernatant of monocytes cultured for 16 h in the presence (gray bar) of selenium (2 µg/ml; mean values for OD were 3.07 vs. 1.34 for untreated; n=15; *, P<0.001). (D) Representative Western blot, which shows sL-selectin from the supernatant of cultured monocytes in the presence (+) or absence (–) of selenium (2 µg/ml).

The question of whether selenium may affect the monocyte activation status was addressed with the single-chain antibody MAN-1, which specifically binds to the activated membrane-activated complex-1 (CD11b/CD18, {alpha}Mβ2) receptor (see Supplemental Methods). Incubation of monocytes with selenium (2 µg/ml) did not result in monocyte activation (see online Supplemental Fig. 1A).

Decreased L-selectin expression occurs after 16 h of incubation with selenium in isolated, cultured monocytes
We assessed L-selectin expression on the surface of isolated and cultured monocytes after 2, 6, and 16 h of incubation in the presence or absence of selenium (2 µg/ml) by flow cytometry. L-selectin expression was unaffected after 2 and 6 h of treatment with selenium. Incubation with selenium for up to 16 h led to a significant decrease in L-selectin surface expression (Fig. 1B) . This observation led us to further characterize whether L-selectin expression is down-regulated by internalization or cleavage of the receptor from the cell surface, which can be measured indirectly by the determination of sL-selectin.

Increased sL-selectin in the supernatant of cultured monocytes after selenium treatment
Incubation of isolated monocytes from healthy donors in the presence of selenium (2 µg/ml) over a period of 16 h led to increased sL-selectin in the cell culture supernatant (Fig. 1C) . The sL-selectin was detected by ELISA. The mean OD in the supernatant from untreated monocytes was 1.34 compared with 3.07 in the supernatant from selenium-treated monocytes (n= 15; P<0.001). The increase in sL-selectin was confirmed by Western blotting of the conditioned media from selenium-treated and untreated monocytes, respectively (Fig. 1D) . Thus, selenium seems to increase L-selectin shedding from monocytes.

L-selectin shedding is dependent on the functional activity of ADAM17/TACE
As ADAM17/TACE is involved in the shedding of L-selectin, we hypothesized that selenium induces L-selectin shedding via a direct stimulatory effect on monocytic-shedding enzymes. As MPs of the ADAM family have been implicated in L-selectin shedding, isolated monocytes were treated with selenium (2 µg/ml) in the absence or presence of TAPI-2 (100 µM), which is a potent ADAM17/TACE inhibitor. TAPI-2 reduced selenium-induced shedding of L-selectin by 68% (n=7; Fig. 2A ; OD selenium treated 2.02 vs. 1.05 selenium and TAPI-2, untreated 0.60). As a positive control, L-selectin shedding was induced by stimulation with PMA (0.25 µg/ml), which is known to induce MMP-dependent shedding of L-selectin. This process was abolished completely by the broad-range MP inhibitor GM6001 (100 µM; Fig. 2B ). For further characterization of the role of ADAM17/TACE in selenium-induced shedding, we used the ADAM10- and ADAM17-specific synthetic hydroxamate MP inhibitors GI254023X and GW280264X. The compound GI254023X has a 100-fold higher potency for the inhibition of ADAM10 as compared with ADAM17, whereas GW280264X inhibits both MPs equally with high potency [24 ]. There was no significant decrease in L-selectin shedding when monocytes were treated with selenium in the presence of the more ADAM10-specific inhibitor GI254023X (10 µM; see Go Fig. 4B ). In the presence of GW280264X (10 µM), selenium-induced L-selectin shedding was decreased significantly by 70% (n=4; see Fig. 4B ; OD selenium treated 0.18 vs. 0.11 selenium+GW280264X-treated, untreated 0.08). These findings strongly argue that ADAM17/TACE mediates selenium-induced L-selectin shedding from monocytes.


Figure 2
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  		Figure 2. Selenium-induced L-selectin shedding is MMP-dependent. (A) Isolated monocytes were cultured for 16 h in the presence (gray and striped bars) or absence (open bar) of selenium (2 µg/ml) and the ADAM17/TACE-specific MMP inhibitor TAPI-2 (100 µM). sL-selectin in the supernatant was determined by ELISA (mean values for OD were 2.02 for selenium vs. 1.05 for selenium+TAPI-2; n=7; *, P=0.03). (B) Isolated monocytes were cultured for 15 min in the presence (plaid and striped bars) or absence (open bar) of PMA (100 ng/ml) and the broad-band MMP inhibitor GM6001 (100 µM). sL-selectin in the supernatant was determined by ELISA, and mean values for OD are depicted. (C) Isolated monocytes were incubated with a fluorogenic TACE substrate, which is cleaved by ADAM17/TACE, in the presence (gray bar) or absence (open bar) of selenium (2 µg/ml; mean values for fluorescence emission at 39 nm were 2115 for untreated vs. 2404; n=14; *, P<0.001). (D) Representative flow cytometry histograms, which show the expression of membrane-bound ADAM17/TACE on isolated monocytes after 16 h of culture in the presence (+) or absence (–) of selenium (2 µg/ml). Gray histograms represent unspecific binding of anti-mouse IgG1–FITC (isotype control); black histograms represent the binding of the FITC-labeled, ADAM17/TACE-specific mAb.


Figure 3
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  		Figure 3. Selenium supplementation causes decreased monocyte rolling and adhesion. Isolated monocytes were cultured in the presence or absence of selenium (2 µg/ml) and thereafter, perfused over a matrix coated with Sialyl-Lewisa at a low shear rate (0.5 dyne/cm2) for 5 min followed by a high shear rate (15 dyne/cm2) for 30 s. (A) Rolling monocytes—untreated (open bar); selenium-treated (gray bar); and selenium + GM6001-treated (striped bar)—were counted in randomly selected 5 s frames (mean values of rolling cells were 7.3 for untreated vs. 3.6 for selenium-treated; n=9; P<0.001). Rolling was partially restored in the additional presence of the broad-band MMP inhibitor GM6001 (striped bar, mean value 4.7; P=0.01 for gray bar vs. striped bar; *, P<0.01; §, P=0.01). (B) Adherent monocytes—untreated (dashed line); selenium-treated (black line); and selenium + GM6001-treated (gray line)—were counted in randomly selected field; each point represents the mean value of nine different experiments.


Figure 4
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  		Figure 4. Selenium-induced shedding occurs in vivo, and ADAM17 is the major "sheddase." (A) Mouse sL-selectin was detected by ELISA in the serum derived from saline (open bar) or selenium-treated (gray bar) C57BL6 mice. The mean values (n=3 per group) were 1146 ± 21 ng/ml for saline treatment and 1310 ± 94 ng/ml for selenium treatment, respectively; *, P < 0.01. (B) Isolated monocytes were cultured for 16 h in the absence (open bar) or presence (all other bars) of selenium (2 µg/ml) and the synthetic hydroxamate MP inibitors GI254023X and GW280264X (both 1 and 10 µM). Both inhibitors block ADAM10 and ADAM17. However, GI254023X has a 100-fold higher potency for the inhibition of ADAM10. sL-selectin in the supernatant was determined by ELISA (mean values for OD were 0.18 for selenium-treated vs. 0.11 for selenium+GW280264X-treated; n=4; *, P=0.024).

Regulation of monocytic ADAM17/TACE by selenium
Flow cytometry of isolated monocytes for ADAM17/TACE expression was performed to determine whether the observed selenium-induced L-selectin shedding occurs as a consequence of up-regulation of membrane-bound ADAM17/TACE. After 16 h of incubation, no difference in the level of ADAM17/TACE expression was found between untreated and selenium-treated monocytes (Fig. 2D) .

For the direct evaluation of selenium’s effects on ADAM17/TACE activity, we used an enzyme substrate assay. Isolated monocytes were incubated with TACE substrate in the presence or absence of selenium. The presence of selenium led to increased TACE substrate turnover by 13.7% (n=14; P<0.001) compared with the untreated control (Fig. 2C) . Although there was a relatively high basal proteolysis of the substrate by untreated cells, these data indicate that the activity of membrane-bound ADAM17/TACE is up-regulated in the presence of selenium.

In an attempt to determine whether the relatively small increase in ADAM17/TACE activity would also lead to increased proteolysis of other ADAM17/TACE substrates, we determined TNF-{alpha} levels in the conditioned media from selenium-treated and nontreated monocytes (see Supplemental Methods). There was no significant increase in the level of TNF-{alpha} in the selenium-treated monocytes. Additionally, basal proteolysis of TNF-{alpha} was inhibited in the presence of 10 µM GW280264X but not GI254023X (see online Supplemental Fig. 1B). This indicates that selenium-induced L-selectin shedding is rather more specific than a result of a general increased activity of ADAM17/TACE.

Selenium-induced L-selectin shedding causes decreased rolling and adhesion under conditions of low shear stress
To evaluate the functional consequences of selenium-induced L-selectin shedding, we performed cell adhesion assays under flow conditions. Isolated monocytes were incubated in the absence or presence of selenium (2 µg/ml) over a period of 16 h. Thereafter, 8 x 104 monocytes/ml were perfused over a Sialyl-Lewisa-coated surface in a parallel plate flow chamber under conditions of low shear stress (0.5 dyne/cm2). The shear stress was increased to a high shear stress at the end of the perfusion time (5 min). The number of rolling monocytes was determined as the mean value from video frames of 5 s duration at four different time-points, each of them 1 min apart. The mean value of rolling monocytes was reduced from 7.3 to 3.6 in the selenium-treated group (Fig. 3A , open and gray bars; P<0.001). Incubation in the presence of the broad-range MMP inhibitor GM6001 partially restored the number of rolling monocytes toward the untreated control group mean (Fig. 3A , striped bar).

The adhesion of monocytes on the Sialyl-Lewisa-coated matrix was then assessed at 30 s, 1:30, 2:30, 3:30, and 4:30 min during the perfusion at low shear stress (0.5 dyne/cm2) and after 30 s of perfusion, using high shear stress (15 dyne/cm2). Compared with the untreated control, adhesion of selenium-treated monocytes was decreased dramatically (Fig. 3B) . Incubation in the presence of GM6001 nearly restored levels of adhesion to the untreated control group mean (Fig. 3B) . The decline of adherent monocytes after application of high shear stress was nearly equal in all groups. Thus, selenium has a significant impact on monocyte adhesion under flow conditions. Overall, there is strong evidence that ADAM17/TACE-induced L-selectin shedding is a major contributor to this effect.

i.v. injection of selenium causes an increase in sL-selectin in mice
To assess the in vivo relevance of our findings, we examined the effect of selenium bolus injection (2 µg/ml total blood volume) followed by a continuous infusion of 2 µg/60 min over the period of 1 h in 8- to 9-week-old C57BL6 mice. Equal volumes of saline bolus followed by a continuous infusion of saline were used for the control group. Mouse sL-selectin was measured by ELISA in serum samples of the mice. Selenium treatment led to a significant increase in sL-selectin (13%) as compared with the basal sL-selectin levels in the control mice (n=3 per group; 1310 ng/ml vs. 1146 ng/ml; P<0.01; Fig. 4A ). These data strongly support our in vitro findings of selenium-induced, increased L-selectin shedding.


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DISCUSSION
 
Decreased serum selenium levels have been associated with decreased activity of GPXs in patients with SIRS [3 ], and supplementation of selenium has been associated with a rise in GPX activity as well as a reduction in mortality in patients with severe sepsis or septic shock [4 , 5 ]. It has been proposed that the beneficial properties of selenium are a result of an antioxidant effect [5 ]. However, antioxidant therapies such as N-acetylcysteine have been evaluated in smaller-sized clinical trials with controversial results [27 28 29 ] and have thus far failed to be recommended as part of routine therapy in patients with severe sepsis [29 ]. Here, we suggest a novel mechanism by which selenium may act in patients with SIRS.

Incubation of monocytes with selenium for a period of 16 h led to increased shedding of L-selectin, mainly mediated by ADAM17/TACE. Specific blockade of ADAM17/TACE with the MP inhibitor TAPI-2 or GW280264X reduced L-selectin shedding significantly. However, it is likely that aside from ADAM17/TACE, other sheddases are involved in the process. This is supported by our observation that PMA-induced L-selectin shedding was almost completely inhibited by the broad-band MP inhibitor GM6001 in contrast to selenium-induced L-selectin shedding, which was not completely inhibited by GM6001 (data not shown). In a recent report, enhanced L-selectin shedding was described for apoptotic neutrophils in chimeric mice lacking ADAM17/TACE [30 ], suggesting that indeed proteases other than TACE may contribute to this process.

As PMA is also a known stimulator for monocytes, we investigated whether selenium may also have an activating effect on monocytes. For this purpose, monocyte activation was measured using a single-chain antibody (MAN-1) that specifically binds to the activated integrin {alpha}Mβ2 (CD11b/CD18) and therefore, allows a direct measurement of monocyte activation [31 ]. Selenium treatment did not activate monocytes and even partially prevented PMA-induced monocyte activation. Additionally, a TNF-{alpha} ELISA was used to rule out potentially cell-activating effects of selenium and to further determine the specificity of the selenium-induced L-selectin shedding. Selenium treatment did not cause any significant increase in TNF-{alpha} levels in the supernatant of cultured human monocytes, but basal ADAM17-mediated shedding of TNF-{alpha} was inhibited significantly in the presence of GW280264X. This observation, that L-selectin shedding can occur in the absence of cell activation, is in line with a report from Kahn et al. [32 ], who observed L-selectin shedding on neutrophils induced by the calmodulin inhibitor trifluoperazine without any signs of neutrophil activation.

Our data suggest that ADAM17/TACE is the major enzyme mediating selenium-induced L-selectin shedding. We sought to determine whether the increased L-selectin shedding in response to selenium treatment is a result of increased activity or increased expression of membrane-bound ADAM17/TACE. We found no changes in the level of ADAM17/TACE expression between selenium-treated or untreated monocytes. However, a significant increase in the activity of ADAM17/TACE was observed, indicating that ADAM17/TACE activity, rather than up-regulation of expression, mediates selenium-induced shedding of L-selectin from monocytes.

As a functional consequence of selenium-induced L-selectin shedding, we observed decreased rolling of monocytes, perfused at shear rates, corresponding to venous or capillary flow. We also observed that blockade of MMPs with the broad-band inhibitor GM6001 partially restored monocyte rolling. Finally, decreased rolling was associated with decreased adhesion over the observed time periods. The changes in L-selectin surface expression after incubation with selenium translated to an increase of sL-selectin in the supernatant of selenium-treated monocytes (Fig. 1C and 1D) . Therefore, it is possible that the decreased L-selectin expression and the increased sL-selectin in the supernatant contributed to the decreased rolling and adhesion. sL-selectin may act as a molecular buffer between the monocytes and the ligand-coated surface. This hypothesis is supported by a recently published in vitro study that elegantly demonstrated increased rolling velocity of neutrophils as a result of mechanically induced L-selectin shedding [33 ]. Interestingly, in this study, the shear-induced mechanical cleavage could also be overcome with a MP inhibitor. This is similar to our study, despite the fact that we observed L-selectin shedding in response to selenium after an incubation period of only 16 h. Although we observed an increase in sL-selectin in the mouse model after a short infusion time of selenium in our in vitro examinations, shorter incubation periods did not result in significant increases in sL-selectin (data not shown). Therefore, a direct effect of selenium on ADAM17/TACE activity seems unlikely. Indeed, we found no difference in TACE-substrate use, with or without selenium in a cell-free assay, where recombinant ADAM17/TACE was used (data not shown).

Decreased serum levels of sL-selectin are found amongst patients with severe sepsis or trauma and have been linked to increased mortality [21 , 22 ]. In vitro and in vivo data indicate that sL-selectin is biologically active and may prevent leukocyte adhesion to endothelial cells [19 , 20 ]. Therefore, decreased sL-selectin levels may lead to overwhelming recruitment of leukocytes within the microcirculation. Once the local release of cytokines and production of ROS have reached a certain level, there may be severe damage to local tissues, potentially leading to end-organ failure.

We addressed the clinically important question of how selenium supplementation might provide therapeutic benefits for sepsis patients. We found that selenium supplementation leads to increased shedding of L-selectin from isolated monocytes, thereby resulting in increased levels of sL-selectin. As proof of the functional consequence of this effect, we demonstrated impaired rolling and adhesion of selenium-treated monocytes under conditions of shear stress. Furthermore, we were able to demonstrate a selenium-induced increase in serum levels of sL-selectin in a mouse model in vivo. To our knowledge, this is the first report to identify a direct effect on shedding and expression of a monocyte adhesion molecule by selenium and to describe the resulting, functional cellular consequences, which may be an important mechanism by which selenium exerts an anti-inflammatory action in patients with severe sepsis.


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ACKNOWLEDGEMENTS
 
The study was supported by a fellowship from the German Cardiac Society to I. A., the medical faculty of the University of Freiburg, and the National Heart Foundation of Australia.

Received July 27, 2007; revised January 7, 2008; accepted January 30, 2008.


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