Int. J. lmmunopharmac., Vol. 14, No. 8, pp. 1383-1390, 1992. Printed in Great Britain.
0192- 0561/92 $5.00 + .00 Pergamon Press Ltd. ©1992 International Society for Immunopharmacology.
EFFECT OF EBSELEN ON POLYMORPHONUCLEAR LEUKOCYTE ADHESION TO AND MIGRATION THROUGH CYTOKINE-ACTIVATED VASCULAR ENDOTHELIUM ANDREW C. ISSEKUTZ* and NANCY LOPES Department of Pediatrics and Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada (Received 5 March 1991 and in final form 1 June 1992)
Abstract - - Ebselen (PZ51, 2-Phenyl-l, 2-Benzoisoselenazol-3-(2H)-one) is a selinyl organic compound with anti-inflammatory properties. Some of its pharmacological effects are thought to result from its peroxidase activity. Here we examined the effects of Ebselen on polymorphonuclear leukocyte (PMNL) adhesion to umbilical vein endothelium and transendothelial migration in a modified Boyden chamber in which both PMNL-dependent and endothelial-dependent (IL-1, TNFa) PMNL adhesion and migration can be measured. Ebselen was found to dose dependently inhibit the adhesion of PMNL to IL-1 activated endothelium and to inhibit transendothelial PMNL migration induced by IL-1 a, and TNFa with an IC50value of 28/aM. Transendothelial migration induced by the PMNL chemotactic agents C5adesAr, and N-formyl - norleu - leu - phe was also inhibited at slightly higher concentrations. The effect of Ebselen was not on endothelial cell activation but on PMNL activation for adhesion and migration. This effect on PMNL was irreversible for the duration of the assay period (75 min). The results suggest that the anti-inflammatory activity of Ebselen may, in part, be due to direct inhibition of PMNL adhesion to vascular endothelium and transendothelial migration in response to a variety of inflammatory mediators.
The hallmark of the inflammatory response is the adherence of blood leukocytes to vascular endothelium and emigration of leukocytes from blood into the extravascular space across postcapillary venules. This is a complex process involving leukocyte cell surface adhesion molecules, which engage their counter receptors on vascular endothelium, metabolic activation of the leukocyte and its contractile apparatus (reviewed in Pober & Cotran, 1990). It is now well recognized that the vascular endothelium is not a passive by-stander in this process. It may in fact initiate the leukoc y t e - vascular endothelial interaction since vascular endothelium can be activated by cytokines, such as interleukin-1 and t u m o r necrosis factor a (TNFa), to upregulate its expression of leukocyte adhesion molecules such as endothelial leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1), which serve as counterreceptors for the adhesion molecules on leukocytes (Bevilacqua, Stengalin, G i m b r o n e & Seed, 1989; Pober & Cotran, 1990; Springer, 1990). Interaction
of these leukocyte and endothelial adhesion molecules is now known to be a prerequisite for leukocyte, especially polymorphonuclear leukocyte (PMNL), adhesion and emigration into sites of inflammation in vivo and for transendothelial migration in vitro (reviewed in Dana & Arnaout, 1988; Price, Beatty & Corpuz, 1987; Smith, Marlin, Rothlein, T o m a n & Anderson, 1989). We have developed recently an in vitro endothelial cell culture system which allows the quantitation of both P M N L adhesion to umbilical vein endothelium and of P M N L transendothelial migration in response to P M N L chemotactic factors or across IL-1 or T N F a activated endothelium (Morzycki, Sadowska & Issekutz, 1990). This assay lends itself to pharmacological studies. A m a j o r goal of inflammation research is the development of pharmacological agents to control detrimenfal processes and limit tissue injury in inflammatory conditions. Ebselen (PZ51; 2 phenyl1,2-benzoisoselenazol-3-(2H)-one) is an organoselenium heterocycle which has been shown to have anti-
*Author to whom correspondence should be addressed at: Department of Pediatrics, Izaak Walton Killam Hospital for Children, 5850 University Avenue, Halifax, Nova Scotia, Canada B3J 3G9. 1383
1384
A. C. ISSEKUTZand N. LOPES
inflammatory effects. It inhibits inflammation in several animal models including cobra venom paw edema, monoarthritis (Parnham, Leyck, Kuhl, Schalkwijk & van den Berg, 1987; Schalkwijk, van den Berg, van de Putte & Joosten, 1986), alveolitis (Cotgreave, Johansson, Westergren, Mold6us & Brattsand, 1988), and to a lesser extent, carageenan paw edema and adjuvant arthritis (Parnham et al., 1987; Parnham, Leyck, Dereu, Winkelmann & Graf, 1985). Ebselen is unique in that it exhibits glutathione (GSH) peroxidase-like activity (Parnham & Graf, 1987; Wendel, Fausel, Safayhi, Tiegs & Otter, 1984), inhibits lipid peroxidation, and has a cyto-protective action (Miiller, Gabriel & Sies, 1985). It also inhibits leukocyte 5-1ipoxygenase (Safayhi, Tiegs & Wendel, 1985; Parnham et al., 1987), and the glutathione peroxidase-like activity of this compound is beF.eved to be involved in the conversion of LTB4 to its biologically inactive 6-transisomer (Kuhl, Borbe, Fischer, R6mer & Safayhi, 1986). Although there have been numerous reports of the in vivo activity of Ebselen and evaluation of its effects on PMNL and macrophage metabolism in vitro, to our knowledge the effect of this compound on PMNL adhesion and migration has not been reported, in particular in relation to leukocyte-endothelial interactions initiated by the inflammatory cytokines, IL-1 and TNFa. Here we report that Ebselen has a marked inhibitory effect on P M N L adhesion and transendothelial migration, whether induced by PMNL chemotactic factors or by cytokine activated endothelium. These findings may be relevant to the anti-inflammatory actions of this compound.
EXPERIMENTAL PROCEDURES
Growth o f endothelial cells Human umbilical vein endothelial cells (HUVE) were isolated and cultured as described by Jaffe, Nachman, Becker & Minick (1973) using the modifications described previously (Morzycki et al., 1990). Briefly, endothelial cells were isolated from umbilical cords with collagenase (Cooper Biomedical, Mississauga, Ontario) treatment, and grown in RPMI-1640 containing 2 mM L-glutamine, 2-mercaptoethanol, sodium pyruvate, penicillin G / streptomycin (Gibco, Grand Island, NY), 20°7o fetal calf serum (FCS, Hyclone, Logan, UT), 25/ag/ml endothelial cell growth supplement (Collaborative Research, Bedford, MA), and heparin (45/ag/ml) (Sigma Chemical Co., St. Louis, MO). Cells were
cultured in gelatin coated 25-cm 2 culture flasks (Nunc, Gibco) in 507o COz-9507o humidified air at 37°C. The cells had typical cobblestone morphology and >98°7o showed staining for factor VIII antigen by immunofluorescence. At confluence HUVE were detached using 0.025°7o trypsin/0.01°/0 Versene (MA Bioproducts, Walkerville, MD) and cultured on PVP-free polycarbonate filters bearing 3 lam pores in Transwell culture plate inserts (6.5 mm diameter, Transwell 3415; Costar, Cambridge, MA). The filters were prepared by coating with 2°7o gelatin followed by 3/ag of human fibronectin (Collaborative Research) in 45 ~1 water and allowed to dry as described previously (Morzycki et al., 1990). HUVE at 2 × 104 cells from the first or second passage were added to the cups above the filter in 0.1 ml complete medium and 0.6 ml medium was added to the lower compartment beneath the filter. The HUVE formed a tight permeability barrier in 6 days. Medium was exchanged for fresh medium 1 - 2 days before (usually day 4 or 5) the monolayers were used for migration studies. The functional integrity of the HUVE monolayer was evaluated using diffusion of ~25I-labelled endotoxin-free human serum albumin (HSA, Connaught Labs, Toronto, Ontario) as described previously (Morzycki et al., 1990). An acceptable endothelium barrier was considered to restrict HSA diffusion to 25%).
Preparation o f P M N L suspension and P M N L adhesion and migration Leukocyte preparation was as described previously (Morzycki et al., 1990). Briefly, peripheral venous blood from healthy human individuals was collected into heparin (5 U / m l blood), acid citrate dextrose (ACD formula A) and red cells were separated by sedimentation with 6% d e x t r a n - s a l i n e solution (Travenol, Malton, Ontario). The leukocyte rich plasma was harvested. Leukocytes and residual red cells were centrifuged (150 g, 10 min) into a pellet and resuspended in Ca 2~, Mg2+-free Tyrode's solution with 5% autologous platelet-poor plasma (PPP) and labelled with 5~Cr sodium chromate (50/aCi/ml) (Amersham Corp, Oakville, Ontario). Labelled PMNLs were separated by centrifugation (300 g, 30 min) on discontinuous (62%/73°70) 10% PPP-Percoll (Pharmacia Fine Chemicals, Dorval, Quebec) gradients. After two washes with Tyrode's solution (Ca 2+, MgZ+-free)-0.5% HSA, the PMNL were resuspended at a final concentration of 3 × 106/ml in RPMI-1640 containing 10 mM
Effect of Ebselen on PMNL Adhesion HEPES and 0.5 07oendotoxin-free HSA. This method yielded >9507o pure PMNL with essentially no red cell contamination and >9507o viability. For assays, HUVE monolayers on the filters and the lower compartment were washed with RPMI-1640 and medium exchanged for RPMI-1640-10070 FCS, 10 mM HEPES. When I L - l a or TNFa were used to stimulate the HUVE, they were added to the lower compartment and incubated for 2 - 3 h at 37°C in a humidified incubator with 507o CO2. Following preincubation with cytokines, the upper and lower surfaces of the filter cup were washed with RPMI-1640 and then transferred to a new macrowell (lower compartment). To this well 0.6 ml of RPMI1640/HEPES/0.5% HSA was added and before immersion of the filter cup, 0.1 ml of [5'Cr]PMNL suspended in the same medium was added to the upper compartment (cup). When PMNL chemotactic factors were used, these were added to the lower chamber at time of PMNL addition to the upper chamber above the HUVE monolayer. After 15 or 75 min incubation, migration was stopped by vigorous washing of the upper compartment twice with 0.1 ml of RPMI-1640 to remove non-adherent PMNLs. The undersurface of the filter was then rinsed with 2 ml of ice-cold PBS/0.2% EDTA solution and collected into the lower compartment. The filter cup was then placed into 0.7 ml of 0.5 M NaOH to allow dissolution of adhered PMNLs. The PMNLs which migrated into the lower compartment and were detached from the filter were lysed by the addition of 0.5% Triton X-100. The NaOH solution bathing the filters (adhered PMNL-51Cr) and the contents of the lower chamber (medium + P B S - EDTA wash of filter undersurface) (migrated PMNL) were analysed for 51Cr and the results are expressed as percent in each fraction of total [51Cr] PMNL added. The "spontaneous" release of 5~Cr from the labelled PMNL was
0192- 0561/92 $5.00 + .00 Pergamon Press Ltd. ©1992 International Society for Immunopharmacology.
EFFECT OF EBSELEN ON POLYMORPHONUCLEAR LEUKOCYTE ADHESION TO AND MIGRATION THROUGH CYTOKINE-ACTIVATED VASCULAR ENDOTHELIUM ANDREW C. ISSEKUTZ* and NANCY LOPES Department of Pediatrics and Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada (Received 5 March 1991 and in final form 1 June 1992)
Abstract - - Ebselen (PZ51, 2-Phenyl-l, 2-Benzoisoselenazol-3-(2H)-one) is a selinyl organic compound with anti-inflammatory properties. Some of its pharmacological effects are thought to result from its peroxidase activity. Here we examined the effects of Ebselen on polymorphonuclear leukocyte (PMNL) adhesion to umbilical vein endothelium and transendothelial migration in a modified Boyden chamber in which both PMNL-dependent and endothelial-dependent (IL-1, TNFa) PMNL adhesion and migration can be measured. Ebselen was found to dose dependently inhibit the adhesion of PMNL to IL-1 activated endothelium and to inhibit transendothelial PMNL migration induced by IL-1 a, and TNFa with an IC50value of 28/aM. Transendothelial migration induced by the PMNL chemotactic agents C5adesAr, and N-formyl - norleu - leu - phe was also inhibited at slightly higher concentrations. The effect of Ebselen was not on endothelial cell activation but on PMNL activation for adhesion and migration. This effect on PMNL was irreversible for the duration of the assay period (75 min). The results suggest that the anti-inflammatory activity of Ebselen may, in part, be due to direct inhibition of PMNL adhesion to vascular endothelium and transendothelial migration in response to a variety of inflammatory mediators.
The hallmark of the inflammatory response is the adherence of blood leukocytes to vascular endothelium and emigration of leukocytes from blood into the extravascular space across postcapillary venules. This is a complex process involving leukocyte cell surface adhesion molecules, which engage their counter receptors on vascular endothelium, metabolic activation of the leukocyte and its contractile apparatus (reviewed in Pober & Cotran, 1990). It is now well recognized that the vascular endothelium is not a passive by-stander in this process. It may in fact initiate the leukoc y t e - vascular endothelial interaction since vascular endothelium can be activated by cytokines, such as interleukin-1 and t u m o r necrosis factor a (TNFa), to upregulate its expression of leukocyte adhesion molecules such as endothelial leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1), which serve as counterreceptors for the adhesion molecules on leukocytes (Bevilacqua, Stengalin, G i m b r o n e & Seed, 1989; Pober & Cotran, 1990; Springer, 1990). Interaction
of these leukocyte and endothelial adhesion molecules is now known to be a prerequisite for leukocyte, especially polymorphonuclear leukocyte (PMNL), adhesion and emigration into sites of inflammation in vivo and for transendothelial migration in vitro (reviewed in Dana & Arnaout, 1988; Price, Beatty & Corpuz, 1987; Smith, Marlin, Rothlein, T o m a n & Anderson, 1989). We have developed recently an in vitro endothelial cell culture system which allows the quantitation of both P M N L adhesion to umbilical vein endothelium and of P M N L transendothelial migration in response to P M N L chemotactic factors or across IL-1 or T N F a activated endothelium (Morzycki, Sadowska & Issekutz, 1990). This assay lends itself to pharmacological studies. A m a j o r goal of inflammation research is the development of pharmacological agents to control detrimenfal processes and limit tissue injury in inflammatory conditions. Ebselen (PZ51; 2 phenyl1,2-benzoisoselenazol-3-(2H)-one) is an organoselenium heterocycle which has been shown to have anti-
*Author to whom correspondence should be addressed at: Department of Pediatrics, Izaak Walton Killam Hospital for Children, 5850 University Avenue, Halifax, Nova Scotia, Canada B3J 3G9. 1383
1384
A. C. ISSEKUTZand N. LOPES
inflammatory effects. It inhibits inflammation in several animal models including cobra venom paw edema, monoarthritis (Parnham, Leyck, Kuhl, Schalkwijk & van den Berg, 1987; Schalkwijk, van den Berg, van de Putte & Joosten, 1986), alveolitis (Cotgreave, Johansson, Westergren, Mold6us & Brattsand, 1988), and to a lesser extent, carageenan paw edema and adjuvant arthritis (Parnham et al., 1987; Parnham, Leyck, Dereu, Winkelmann & Graf, 1985). Ebselen is unique in that it exhibits glutathione (GSH) peroxidase-like activity (Parnham & Graf, 1987; Wendel, Fausel, Safayhi, Tiegs & Otter, 1984), inhibits lipid peroxidation, and has a cyto-protective action (Miiller, Gabriel & Sies, 1985). It also inhibits leukocyte 5-1ipoxygenase (Safayhi, Tiegs & Wendel, 1985; Parnham et al., 1987), and the glutathione peroxidase-like activity of this compound is beF.eved to be involved in the conversion of LTB4 to its biologically inactive 6-transisomer (Kuhl, Borbe, Fischer, R6mer & Safayhi, 1986). Although there have been numerous reports of the in vivo activity of Ebselen and evaluation of its effects on PMNL and macrophage metabolism in vitro, to our knowledge the effect of this compound on PMNL adhesion and migration has not been reported, in particular in relation to leukocyte-endothelial interactions initiated by the inflammatory cytokines, IL-1 and TNFa. Here we report that Ebselen has a marked inhibitory effect on P M N L adhesion and transendothelial migration, whether induced by PMNL chemotactic factors or by cytokine activated endothelium. These findings may be relevant to the anti-inflammatory actions of this compound.
EXPERIMENTAL PROCEDURES
Growth o f endothelial cells Human umbilical vein endothelial cells (HUVE) were isolated and cultured as described by Jaffe, Nachman, Becker & Minick (1973) using the modifications described previously (Morzycki et al., 1990). Briefly, endothelial cells were isolated from umbilical cords with collagenase (Cooper Biomedical, Mississauga, Ontario) treatment, and grown in RPMI-1640 containing 2 mM L-glutamine, 2-mercaptoethanol, sodium pyruvate, penicillin G / streptomycin (Gibco, Grand Island, NY), 20°7o fetal calf serum (FCS, Hyclone, Logan, UT), 25/ag/ml endothelial cell growth supplement (Collaborative Research, Bedford, MA), and heparin (45/ag/ml) (Sigma Chemical Co., St. Louis, MO). Cells were
cultured in gelatin coated 25-cm 2 culture flasks (Nunc, Gibco) in 507o COz-9507o humidified air at 37°C. The cells had typical cobblestone morphology and >98°7o showed staining for factor VIII antigen by immunofluorescence. At confluence HUVE were detached using 0.025°7o trypsin/0.01°/0 Versene (MA Bioproducts, Walkerville, MD) and cultured on PVP-free polycarbonate filters bearing 3 lam pores in Transwell culture plate inserts (6.5 mm diameter, Transwell 3415; Costar, Cambridge, MA). The filters were prepared by coating with 2°7o gelatin followed by 3/ag of human fibronectin (Collaborative Research) in 45 ~1 water and allowed to dry as described previously (Morzycki et al., 1990). HUVE at 2 × 104 cells from the first or second passage were added to the cups above the filter in 0.1 ml complete medium and 0.6 ml medium was added to the lower compartment beneath the filter. The HUVE formed a tight permeability barrier in 6 days. Medium was exchanged for fresh medium 1 - 2 days before (usually day 4 or 5) the monolayers were used for migration studies. The functional integrity of the HUVE monolayer was evaluated using diffusion of ~25I-labelled endotoxin-free human serum albumin (HSA, Connaught Labs, Toronto, Ontario) as described previously (Morzycki et al., 1990). An acceptable endothelium barrier was considered to restrict HSA diffusion to 25%).
Preparation o f P M N L suspension and P M N L adhesion and migration Leukocyte preparation was as described previously (Morzycki et al., 1990). Briefly, peripheral venous blood from healthy human individuals was collected into heparin (5 U / m l blood), acid citrate dextrose (ACD formula A) and red cells were separated by sedimentation with 6% d e x t r a n - s a l i n e solution (Travenol, Malton, Ontario). The leukocyte rich plasma was harvested. Leukocytes and residual red cells were centrifuged (150 g, 10 min) into a pellet and resuspended in Ca 2~, Mg2+-free Tyrode's solution with 5% autologous platelet-poor plasma (PPP) and labelled with 5~Cr sodium chromate (50/aCi/ml) (Amersham Corp, Oakville, Ontario). Labelled PMNLs were separated by centrifugation (300 g, 30 min) on discontinuous (62%/73°70) 10% PPP-Percoll (Pharmacia Fine Chemicals, Dorval, Quebec) gradients. After two washes with Tyrode's solution (Ca 2+, MgZ+-free)-0.5% HSA, the PMNL were resuspended at a final concentration of 3 × 106/ml in RPMI-1640 containing 10 mM
Effect of Ebselen on PMNL Adhesion HEPES and 0.5 07oendotoxin-free HSA. This method yielded >9507o pure PMNL with essentially no red cell contamination and >9507o viability. For assays, HUVE monolayers on the filters and the lower compartment were washed with RPMI-1640 and medium exchanged for RPMI-1640-10070 FCS, 10 mM HEPES. When I L - l a or TNFa were used to stimulate the HUVE, they were added to the lower compartment and incubated for 2 - 3 h at 37°C in a humidified incubator with 507o CO2. Following preincubation with cytokines, the upper and lower surfaces of the filter cup were washed with RPMI-1640 and then transferred to a new macrowell (lower compartment). To this well 0.6 ml of RPMI1640/HEPES/0.5% HSA was added and before immersion of the filter cup, 0.1 ml of [5'Cr]PMNL suspended in the same medium was added to the upper compartment (cup). When PMNL chemotactic factors were used, these were added to the lower chamber at time of PMNL addition to the upper chamber above the HUVE monolayer. After 15 or 75 min incubation, migration was stopped by vigorous washing of the upper compartment twice with 0.1 ml of RPMI-1640 to remove non-adherent PMNLs. The undersurface of the filter was then rinsed with 2 ml of ice-cold PBS/0.2% EDTA solution and collected into the lower compartment. The filter cup was then placed into 0.7 ml of 0.5 M NaOH to allow dissolution of adhered PMNLs. The PMNLs which migrated into the lower compartment and were detached from the filter were lysed by the addition of 0.5% Triton X-100. The NaOH solution bathing the filters (adhered PMNL-51Cr) and the contents of the lower chamber (medium + P B S - EDTA wash of filter undersurface) (migrated PMNL) were analysed for 51Cr and the results are expressed as percent in each fraction of total [51Cr] PMNL added. The "spontaneous" release of 5~Cr from the labelled PMNL was
