SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992
A Role for α-Thrombin in Polymorphonuclear Leukocyte Recruitment During Inflammation
In recent years, there is increasing evidence that thrombin, the key enzyme in the coagulation cascade, may play a role in the development of the inflammatory response. A fundamental component of inflammation is the adherence of polymorphonuclear leukocytes (PMNL) to the vascular endothelium and subsequent migration into the inflamed tissue. Numerous studies have demon strated that α-thrombin is a potent activator of this vascu lar barrier, with effects relevant to inflammation. α-Thrombin stimulates the production by vascular endo thelial cells of prostacyclin,1 thromboplastin,2 tissue plasminogen activator,3 and platelet activator factor (PAF).4 α-Thrombin induces reversible contraction of the endothelial cytoskeleton, resulting in endothelial gap formation and increased endothelial permeability.5'6 The coagulation system is intimately intertwined with the inflammatory response, and there is a growing body of data pointing to the direct participation of throm bin in a variety of disease processes. Leukocytic infiltra tion is regularly associated with thrombotic disorders, such as phlebothrombosis and thrombophlebitis.7 Many forms of vasculitis are characterized by fibrin deposition, thrombi, and leukocyte infiltration of the blood vessel wall.8 Thrombi are a prominent histologic feature in acute proliferative glomerulonephritis.9 The tissue dam age seen in immune reactions such as the experimental Shwartzman reaction10 and hyperacute renal allograft re action is a direct result of activation of coagulation.8 In several forms of chronic inflammation, including rheu matoid arthritis,11 a consistent finding is both leukocyte accumulation and evidence that thrombin was generated either by directly detecting it in the synovial fluid or by
From the Departments of Pediatrics and Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada. Reprint requests: Dr. Issekutz, Department of Pediatrics, Izaak Walton Killam Hospital for Children, 5850 University Avenue, Hali fax, Nova Scotia, B3J 3G9 Canada.
histologic demonstration of fibrin deposition.11 In exper imental animals such as sheep, intravenous thrombin in fusion results in PMNL accumulation in the pulmonary circulation and PMNL-induced pulmonary vascular in jury.12 The sequestration of PMNL in the pulmonary circulation and the oxidant-induced tissue injury that ac companies such accumulation also plays a role in adult respiratory distress syndrome.13 More direct evidence for a role for thrombin in the development of inflammatory processes comes from in vitro studies. α-Thrombin is reported to be a chemotaxin for human monocytes14 and can markedly increase the ability of endothelial cells to adhere both lymphocytes and the monocyte-like U937 cell line.15 PMNLs are one of the major leukocyte types found at sites of inflamma tion and thrombin also has direct effects on PMNLs. Bizios et al16 and more recently Morin et al17 demon strated that thrombin directly induces PMNL chemotaxis. It has also been demonstrated that thrombin stimu lates PMNL de novo synthesis and secretion of tissue kallikrein (TK) activity, thus providing a mechanism for PMNL to deliver TK to a site of inflammation.18 To reach a site of tissue injury or infection, circulat ing PMNL must first marginate along the blood vessel endothelium and form stable adhesions before migrating into the extravascular space. Thrombin has been reported to elicit two temporally different effects on PMNL-endothelial interactions. Several previous studies have re ported that thrombin could act directly on endothelial cells to increase their adhesiveness for PMNL. 19,20 In the first study, maximal adhesion of PMNL occurred within 5 minutes of incubation of endothelial cells with α-thrombin.19 This type of adhesion was further charac terized to be strictly limited to the endothelial cell surface with no sign of further migration.21 This rapidly induced endothelial adhesiveness appears due to mobilization of intracellular stores of the adhesion glycoprotein GMP140 to the endothelial surface22 and to the synthesis of
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York; NY 10016. All rights reserved.
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PAF by the endothelium.4 The counter ligands on PMNL, which may bind to these endothelial molecules, are thought to involve, in part, the glycoprotein LECAM-1 (Mel-14) and the PAF receptor. 19,23,24,25 In a study by Bizios et al, 20 which used an ovine system, maximal stimulation of PMNL adhesion occurred when the endothelial cells were pretreated with α-thrombin for at least 60 minutes. This type of late-phase interaction was shown to lead subsequently to subendothelial infil tration, following a 6-hour pretreatment with α-throm bin.21 The mechanistic relationship between the rapid and these delayed effects of thrombin on PMNL-endothelial interactions is not yet clear. It is known that cytokines such as interleukin-1 (IL1α) and tumor necrosis factor (TNFα) induce de novo synthesis, by vascular endothelium, of an endothelial leukocyte adhesion molecule (ELAM-1) and up-regulation of the expression of intercellular adhesion mole cule-1 (ICAM-1).26,27 Both of these molecules contrib ute not only to PMNL adhesion, but also to PMNL transendothelial migration in vitro. 28,29 These proinflam matory cytokines also stimulate endothelial cell surface expression of tissue procoagulant or thromboplastin30,31 and down-regulate anticoagulant factors such as throm bomodulin.32 As a result, endothelium involved in in flammation likely promotes blood coagulation. Because of the increasing potential for interactions between blood coagulation and inflammation, the goal of our investiga tions has been to determine the effect of α-thrombin on PMNL interaction with cytokine-activated endothelium, as is likely to occur at sites of inflammation.
MATERIALS AND METHODS Endothelial cells from human umbilical veins (HUVE) were harvested and cultured by the method of Jaffe et al 33 using modifications previously described.34 At confluence, HUVE were trypsinized and isolated cells were grown for 6 days at 37°C to form a tight monolayer on gelatin/fibronectin-coated polycarbonate filters (3 |xm pores) attached to culture plate inserts (Costar, Cam bridge, MA). Using this system, PMNL adherence and transendothelial migration, utilizing 51Cr-labeled human PMNL, can be separately measured, as we previously reported.35 Monolayer permeability was measured using 125 I-albumin. For assays, HUVE monolayer filters were stimu lated for 3 hours from the basal direction (lower chamber) with IL-1α or TNFα in 0.6 ml of RPMI-1640 10% fetal calf serum (FCS). This stimulation was followed by a media change and to the lower chamber 0.6 ml of RPMI1640 0.5% human serum albumin (HSA)-N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) (10
mM) was added. Purified 51Cr-labeled human PMNL (2.5 x 105/0.1 ml) in RPMI-1640-HSA-HEPES contain ing 3-5 x 104 cpm 125I-HSA was added to the chamber above the monolayer/filter unit. At this point, α-throm bin was added, usually to the upper chamber, unless otherwise indicated, and the chemotactic factor f-norLeu-Leu-Phe (FNLP), when used, was added to the chamber below the monolayer. The PMNL and HUVE were incubated at 37°C for 15 to 90 minutes, migration was stopped by washing the surface above the monolayer to remove nonadherent PMNL, and the undersurface of the filter was rinsed and collected into the lower compart ment. The percent of applied 51Cr-PMNL remaining ad herent to the monolayer, the percent that migrated into the lower chamber beneath the monolayer/filter unit, and the percent of 125I-HSA added to the upper chamber that diffused across the monolayer into the lower chamber were determined by a gamma spectrometer. In vivo studies were carried out on a rabbit dermal inflammation model. New Zealand White rabbits of ei ther sex weighing 3 to 4 kg were shaved and 40 to 50 skin sites were designated for intradermal injection. The quantitation of leukocyte accumulation in dermal inflam matory reactions was performed by isolation of rabbit blood leukocytes, labeling them with 51Cr and intrave nous infusion according to previously published meth ods. 36,37 Inflammatory stimuli, including IL-1α, TNFα, NAP-1 (IL-8), zymosan-activated serum (ZAS, contain ing C5a desArg ), 36,38 or control 0.1% HSA phosphatebuffered saline (PBS) were injected intradermally im mediately following intravenous infusion of labeled leu kocytes. Sixty minutes later, at the peak of PMNL migra tion, α-thrombin was injected intradermally into the same and new sites together with intravenous injection of 125 I-HSA. Then, 30 minutes later, after a total of 90 minutes, the animal was sacrificed, the skin removed, and the lesions punched out. The accumulation of 51Cr (on leukocytes) and 125I (on albumin) in the tissue was determined by a gamma spectrometer. The 51Cr radioiso tope content was converted to the number of PMNLs (x 106) accumulated in the lesions during the reaction as previously reported,36 and the 125I content indicating plasma protein leakage was converted to microliters of plasma per site, based on plasma 125I content at sacrifice, as reviewed previously.37 An enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of ELAM-1 and ICAM-1 on the endothelial cell surface, as previously described.35 Briefly, HUVE was grown to confluence in microwells and then stimulated with or without IL-1α for 3 hours. The monolayers were rinsed once with RPMI, and subsequently stimulated with α-thrombin for 10 min utes. Following another rinse with RPMI-1640, mono clonal antibodies H4/18 (anti-ELAM-1), RR 1/1 (anti-
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ICAM-1), and 3H11B9 (antipertussis toxin as negative control) at 1:1000 of ascites were applied in RPMI-1640, 5% FCS with 0.1% azide for 60 minutes at 37°C. These antibodies were removed, the monolayer washed, and then incubated with a 1:3000 dilution of peroxidase con jugated goat antimouse immunoglobulin G (IgG) in RPMI-1640, 5% FCS for 60 minutes at 37°C. Unbound conjugate was removed, and the enzyme substrate, O-phenylenediamine (0.34 mg/ml) in a pH 5 citratephosphate buffer, was then added. Color development was stopped with 2 M sulfuric acid and the optical den sity of each well read at 492 nm in a Biotek ELISA plate reader.
RESULTS Effect of Thrombin on Polymorphonuclear Leukocyte Migration in Vitro Previous studies have shown that α-thrombin can elicit two temporally different forms of PMNL-endothelial interactions.19,21 In our study, α-thrombin (0.1 U/ml) induced rapid, transient, but relatively weak (3.4% ± 1.4 versus control of 0.3 ± 0.1%) adherence of added PMNLs during 15 to 30 minutes of PMNL incuba tion with endothelium. In contrast, IL-1α (0.05 ng/ml) induced adherence of 21.2% ± 2.0 of the PMNL (not shown). Figure 1 shows the time course of PMNL migra tion through unstimulated endothelial monolayers and monolayers treated with α-thrombin (0.1 U/ml) added to the upper chamber (apical surface) at the time of PMNL addition. As shown in Figure 1, α-thrombin stimulation of endothelium was capable of inducing transendothelial
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migration of 13.2% ± 0.62 of the PMNL by 15 minutes, which plateaued between 30 and 90 minutes. A mean PMNL migration of 8.4% ± 0.92 at a 30-minute incuba tion time was seen in 15 other experiments in which α-thrombin (0.1 U/ml) was used to stimulate the endo thelium. This migration did not increase significantly at longer incubation times, indicating the thrombin effect was short-lived for PMNL migration. α-Thrombin-induced PMNL migration was relatively low when com pared with that observed across IL-1α stimulated endo thelium, in which case PMNL migration continued to increase with incubation up to 90 minutes, as shown in Figure 1 and reported previously.35 Very few PMNLs migrated through unstimulated endothelium (less than 5%) at any time. Consistent with observations by other workers, α-thrombin exerted its migration effects via a direct ac tion on the endothelium,21'39 because in the assay system used, α-thrombin was added to the endothelium for 10 minutes, the monolayer washed, and PMNLs subse quently added, so α-thrombin was not available to inter act with the PMNLs. There was no significant difference in the response whether the endothelium was stimulated via the apical surface (thrombin added to upper chamber above monolayer) or basal surface (lower chamber below monolayer; not shown). In this system, α-thrombin did not have a direct PMNL chemotactic effect or induce PMNL migration across bare membranes in the absence of endothelium (not shown). Using the described system, we have previously shown that pretreatment of endothelial monolayers with IL-1α for 2 to 4 hours is optimal for activation of the endothelium for PMNL adherence and migration.35 In view of evidence that α-thrombin shares with IL-1α the
FIG. 1. Kinetics of the enhancement by α-thrombin of interleukin-1a (IL1α)-induced polymorphonuclear leu kocyte (PMNL) transendothelial mi gration. Human umbilical vein endothelial monolayers were unstim ulated, prestimulated for 3 hours with 0.05 ng/ml IL-1α, or stimulated 10 min utes with 0.1 U/ml α-thrombin. PMNLs were added and incubated to allow mi gration for varying periods of time. Values are the percent of added PMNLs that migrated across the monolayer/filter unit. Means ± SD of triplicate determinations are shown. Similar results were obtained in four other time-course experiments. An as terisk denotes significant (p < 0.05) en hancement over additive values.
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ability to activate endothelial cells, we studied the effect of α-thrombin stimulation on endothelium prestimulated with IL-1α, to investigate whether α-thrombin may mod ulate this type of PMNL-endothelial interaction. Figure 1 shows that α-thrombin was able to alter the kinetics of the IL-1 induced migration by increasing, in a synergistic manner, the initial rate of PMNL transendothelial migra tion. At both 15 and 30 minute time points, the migration was enhanced by 58% over additive values. At the later time points, the migration of PMNLs across IL-1α acti vated endothelium, in the absence of thrombin, caught up to the absolute values seen with thrombin addition. We then examined the effect of α-thrombin on PMNL migration across HUVE monolayers induced by TNFα and the synthetic PMNL chemotactic factor FNLP. TNFα is known to activate endothelium for adhe siveness for PMNL analogous to IL-1α, 40 and this also results in PMNL transendothelial migration. 21,28,29,35 In contrast to IL-1α and TNFα, FNLP (3 x 10 - 8 M) does not directly activate HUVE, and when added to the lower chamber, it induces PMNL-transendothelial migration by a direct, receptor-mediated chemotactic effect on PMNL. Figure 2 shows the results of an experiment in which the endothelium was treated with α-thrombin (0.1 U/ml) af ter prestimulation with IL-1α (0.05 ng/ml) or TNFα (20 U/ml), or when FNLP was added into the lower chamber. At a 30-minute PMNL migration period, α-thrombin en hanced, in a synergistic manner, the PMNL migration induced by IL-1α and FNLP and had an additive effect on TNFα-induced migration. To determine whether α-thrombin enhanced PMNL migration via its proteolytic activity, experiments were carried out in the same fashion using α-thrombins in which the catalytic site was irreversibly inhibited by diisopropylfluorophosphate (DIP) or D-phenylalanyl-L-
prolyl-L-arginine chloromethyl ketone (PPACK) treat ment.41 Results in Figure 3 show that these enzymatically inactive thrombins did not enhance PMNL transendothe lial migration. Thus, although DIP-thrombin, PPACKthrombin, and α-thrombin are known to bind the same receptor on endothelium with similar affinity,42,43 the findings indicate that the enhanced PMNL migration in duced by α-thrombin is dependent on enzymatic activity and not only receptor binding.
Effect of Thrombin on Polymorphonuclear Leukocyte Migration in Vivo The observations already described were further evaluated in an in vivo rabbit dermal inflammation model. Initially, we examined the effect of various doses of α-thrombin on dermal leukocyte accumulation and found that intradermal injection of α-thrombin at a dose of 10 U/site induced PMNL accumulation of 1.5 x 106 PMNL/site by 90 minutes. This PMNL infiltration was consistently greater than control (0.1% HSA in PBS) values of 0.2 x 106 PMNL/site, but much weaker than responses to IL-1α (2 ng/site) which reached 16 x 106 PMNL/site. We then examined the effect of α-thrombin on the PMNL accumulation induced by a dose range of IL-1α (0.06 to 2 ng/site) injected intradermally. One hour after the intradermal injection of IL-1α (and after the intrave nous infusion of 51Cr-labeled leukocytes), these sites were reinjected intradermally with 10 U/site of α-throm bin or HSA-PBS control. As shown in Figure 4, in vivo findings corroborated the in vitro results. A significant enhancement of PMNL accumulation by α-thrombin oc curred over a wide dose range of IL-1α, demonstrating that this enhancement is not dependent on the IL-1α
FIG. 2. The effect of α-thrombin (0.1 U/ml) on polymorphonuclear leuko cyte (PMNL) transendothelial migra tion in vitro induced by interleukin-1α (IL-1a) (0.05 ng/ml), tumor necrosis factor-alpha (TNFα) (20 U/ml) and FNLP (3 x 10 -8 M). Human umbilical vein endothelial cells (HUVE) were pretreated for 3 hours with IL-1α or TNFα prior to the addition of thrombin, as in Figure 1. FNLP was added to the lower chamber without pretreatment of HUVE at time of thrombin and PMNL addition. Values are mean ± SD of triplicate determinations and are representative of five experiments. An asterisk denotes significant (p < 0.001) enhancement over additive val ues.
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α-thrombin, resulted in synergistically increased PMNL accumulation (190 to 260%), that is, greater than the additive sum of each response alone, which is expressed as the 100% value. In contrast, no accumulation greater than the 100% additive value was observed with NAP-1 and ZAS, the chemotactic agents with α-thrombin com binations. Our findings thus suggest that α-thrombin's effect on the rate of PMNL transmigration induced by IL-1α observed in the in vitro assay is relevant to the dynamics of PMNL adhesion to and migration across microvessels in vivo.
FIG. 3. Effect of enzymatically inactive thrombins on interleukin-1α (IL-1α)-induced polymorphonuclear leukocytes (PMNL) migration. Human umbilical vein endothelial (HUVE) monolayers were stimulated with 33 ng/ml α-thrombin (0.1 U/ml) or 330 ng/ml of active site inhibited thrombins, diisopropylfluorophosphate (DIP) or D-phenylalanyl-L-prolyl-Larginine chloromethylketone (PPACK), 10 minutes prior to PMNL addition. The effect of these thrombins on PMNL re sponses on HUVE prestimulated for 3 hours with 0.05 ng/ml IL-1 is also shown. Data are presented as mean ± SD of tripli cates, representative of three experiments. PMNL migration was for 30 minutes.
dose. Histologic examination of the lesions confirmed these quantitative differences and that the leukocytic in filtrate was more than 90% PMNLs with the PMNLs in the extravascular connective tissue (not shown). There was no sign of thrombosis with the doses of α-thrombin used. α-Thrombin's capacity to affect PMNL accumula tion induced by other agents known to promote PMNL migration was then studied. TNFα (1000 U/ml), another cytokine known to activate endothelial cells, was used in the same manner as IL-1α. NAP-1 (IL-8; 2 µg/site), a newly characterized peptide cytokine known to be highly chemotactic for PMNLs44 was given intradermally 30 minutes prior to the injection of α-thrombin. Rabbit ZAS, the active component of which is known to be C5a desArg , 38 also a PMNL chemotactic factor, was in jected in a 1:50 dilution 60 minutes prior to α-thrombin. Results in Figure 5 show that combinations of IL-1α followed by α-thrombin, and TNFα followed by
Leukocyte adherence to and migration across an endothelial barrier involves interaction between PMNL surface molecules and ligands expressed on the surface of the endothelium, such as ELAM-1 and ICAM-1. 26-29,45 To evaluate the relative involvement of these two ligands in the enhanced migration induced by α-thrombin across IL-1α and TNFα stimulated endothelium, an ELISA was used to measure the expression of these molecules on the endothelial cell surface. As shown in Table 1, there was no significant change in the expression of either of these ligands in response to stimulation with α-thrombin. Thus, the enhanced migration seen in response to α-thrombin stimulation of IL-1α prestimulated endothe lium does not appear to be due to an up-regulation of these ligands on the endothelial cell surface. Receptor-mediated agonists, such as α-thrombin, have been shown to induce rapid mobilization of intracel lular stores of the adhesion glycoprotein GMP-140 to the endothelial surface.22 Structurally similar to ELAM-1, 46 GMP-140 appears to serve as a receptor for PMNL glyco proteins, especially LECAM-1 (Mel 14).23 An additional mechanism, recently recognized to contribute to endo thelial cell-PMNL interactions in response to α-throm bin, is PAF expression on the endothelial cell membrane, likely via PAF receptors on the PMNL. 19,25 To evaluate the contribution of GMP-140 in our observations, eight experiments were performed with blocking anti-GMP140 monoclonal antibody Gl and the nonblocking mAb S12 (as a negative control) to evaluate the effect on the enhancement by α-thrombin of PMNL migration across IL-1α-stimulated endothelium. The HUVE was stimu lated with α-thrombin for 10 minutes in the presence of these monoclonal antibodies before PMNL were added. To evaluate the role of HUVE-generated PAF, four ex periments were also carried out in which PMNL were pretreated with various doses (0.5 to 50 µg/ml) of the PAF antagonists, WEB-2086 or CV3988, before their addition to the HUVE. No consistent inhibition of the
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Investigations of the Mechanism of Enhancement of Polymorphonuclear Leukocytes Migration by α-Thrombin
SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 18, NO. 3, 1992
FIG. 4. α-Thrombin enhancement of interleukin-1α (IL-1α)-induced dermal polymorphonuclear leukocyte (PMNL) accumulation in rabbits. 51Cr-labeled leukocytes were injected intravenously and skin sites received IL-1α in a dose range of 0.06-2 ng/site or 0.1% human serum albumin-phosphate buffered saline (HSA-PBS) control injected intradermally (0.2 ml). Sixty minutes later, the same sites were reinjected intradermally with 0.1 ml of 10 U/site α-thrombin or 0.1% HSA-PBS control. At the time of sacrifice, all lesions were 90 minutes of age. Control (HSA-PBS) injection alone did not induce significant PMNL accumulation (0.2 x 106 PMNL/site). The left hand bar in each pair gives the proportional breakdown of the sum of the thrombin alone and IL-1 alone responses. Values are mean ± SD of triplicate sites, representative of five experiments. An asterisk denotes significant (p < 0.05) enhancement over additive values. All doses and combinations were performed in the same animal.
thrombin enhancement of PMNL migration was ob served using either the anti-GMP-140 monoclonal anti bodies or the PAF antagonists (data not shown). It ap pears that these mechanisms, at least individually, are not responsible for the α-thrombin enhancement of PMNL migration across IL-1 -activated HUVE and complemen tary responses, perhaps with other ligand-receptor sys tems, may be involved.
SUMMARY AND CONCLUSIONS Interactions of leukocytes with the vascular endo thelium is fundamental to many aspects of immune and inflammatory responses. In this report, the ability of α-thrombin, a key enzyme in the coagulation cascade, to alter endothelial cell adhesiveness for PMNL and to stim ulate their transmigration was examined. Both our in vitro and in vivo findings suggest that α-thrombin by itself activates relatively weak endothelial adhesion, as reported by others, and is a weak stimulus for PMNL migration. However, more significantly, our findings have shown that α-thrombin synergistically increases the PMNL transmigration in vitro and PMNL accumulation in vivo induced by IL-1α. This suggests that the en hanced rate of PMNL transmigration observed in vitro with α-thrombin plus IL-1α is relevant to the dynamics of
PMNL adhesion and migration across microvessels in vivo. The results demonstrate an accessory role for α-thrombin-dependent mechanisms, perhaps to stabilize or amplify the adhesion mechanisms induced by IL-1α or TNFα, leading to immobilization of increased numbers of PMNL in the microvessels under shear stress condi tions, and thus result in enhanced PMNL transendothelial migration and tissue infiltration. In recent years, potential proinflammatory actions of α-thrombin have been recognized, especially with re gard to vascular endothelial permeability and leukocyteendothelial interactions. With the growing interest in the potential interactions between inflammation and the co agulation cascade, our results may be relevant to a wide variety of inflammatory reactions in which a procoagulant environment can develop, potentially leading to α-thrombin generation at the endothelial cell surface. Thus, further studies elucidating the in vivo contribution of thrombin in a variety of inflammatory reactions appear warranted. Studies utilizing PAF antagonists in vivo as well as the specific thrombin inhibitor hirudin, to further determine thrombin's role in inflammatory processes, are under way. Acknowledgments: The authors are grateful for gifts of monoclonal antibodies H4/18 from Dr. M. Gimbrone (Harvard Medical School, Boston, MA), RR 1/1 from Dr. T. Springer
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TABLE 1. Effect of α-Thrombin and lnterleukin-1α on Endothelial Cell Expression of ELAM-1 or ICAM-1
HUVE Treatment*
ELAM-1
ICAM-1
Unstimulated
A Role for α-Thrombin in Polymorphonuclear Leukocyte Recruitment During Inflammation
In recent years, there is increasing evidence that thrombin, the key enzyme in the coagulation cascade, may play a role in the development of the inflammatory response. A fundamental component of inflammation is the adherence of polymorphonuclear leukocytes (PMNL) to the vascular endothelium and subsequent migration into the inflamed tissue. Numerous studies have demon strated that α-thrombin is a potent activator of this vascu lar barrier, with effects relevant to inflammation. α-Thrombin stimulates the production by vascular endo thelial cells of prostacyclin,1 thromboplastin,2 tissue plasminogen activator,3 and platelet activator factor (PAF).4 α-Thrombin induces reversible contraction of the endothelial cytoskeleton, resulting in endothelial gap formation and increased endothelial permeability.5'6 The coagulation system is intimately intertwined with the inflammatory response, and there is a growing body of data pointing to the direct participation of throm bin in a variety of disease processes. Leukocytic infiltra tion is regularly associated with thrombotic disorders, such as phlebothrombosis and thrombophlebitis.7 Many forms of vasculitis are characterized by fibrin deposition, thrombi, and leukocyte infiltration of the blood vessel wall.8 Thrombi are a prominent histologic feature in acute proliferative glomerulonephritis.9 The tissue dam age seen in immune reactions such as the experimental Shwartzman reaction10 and hyperacute renal allograft re action is a direct result of activation of coagulation.8 In several forms of chronic inflammation, including rheu matoid arthritis,11 a consistent finding is both leukocyte accumulation and evidence that thrombin was generated either by directly detecting it in the synovial fluid or by
From the Departments of Pediatrics and Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada. Reprint requests: Dr. Issekutz, Department of Pediatrics, Izaak Walton Killam Hospital for Children, 5850 University Avenue, Hali fax, Nova Scotia, B3J 3G9 Canada.
histologic demonstration of fibrin deposition.11 In exper imental animals such as sheep, intravenous thrombin in fusion results in PMNL accumulation in the pulmonary circulation and PMNL-induced pulmonary vascular in jury.12 The sequestration of PMNL in the pulmonary circulation and the oxidant-induced tissue injury that ac companies such accumulation also plays a role in adult respiratory distress syndrome.13 More direct evidence for a role for thrombin in the development of inflammatory processes comes from in vitro studies. α-Thrombin is reported to be a chemotaxin for human monocytes14 and can markedly increase the ability of endothelial cells to adhere both lymphocytes and the monocyte-like U937 cell line.15 PMNLs are one of the major leukocyte types found at sites of inflamma tion and thrombin also has direct effects on PMNLs. Bizios et al16 and more recently Morin et al17 demon strated that thrombin directly induces PMNL chemotaxis. It has also been demonstrated that thrombin stimu lates PMNL de novo synthesis and secretion of tissue kallikrein (TK) activity, thus providing a mechanism for PMNL to deliver TK to a site of inflammation.18 To reach a site of tissue injury or infection, circulat ing PMNL must first marginate along the blood vessel endothelium and form stable adhesions before migrating into the extravascular space. Thrombin has been reported to elicit two temporally different effects on PMNL-endothelial interactions. Several previous studies have re ported that thrombin could act directly on endothelial cells to increase their adhesiveness for PMNL. 19,20 In the first study, maximal adhesion of PMNL occurred within 5 minutes of incubation of endothelial cells with α-thrombin.19 This type of adhesion was further charac terized to be strictly limited to the endothelial cell surface with no sign of further migration.21 This rapidly induced endothelial adhesiveness appears due to mobilization of intracellular stores of the adhesion glycoprotein GMP140 to the endothelial surface22 and to the synthesis of
Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York; NY 10016. All rights reserved.
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PAF by the endothelium.4 The counter ligands on PMNL, which may bind to these endothelial molecules, are thought to involve, in part, the glycoprotein LECAM-1 (Mel-14) and the PAF receptor. 19,23,24,25 In a study by Bizios et al, 20 which used an ovine system, maximal stimulation of PMNL adhesion occurred when the endothelial cells were pretreated with α-thrombin for at least 60 minutes. This type of late-phase interaction was shown to lead subsequently to subendothelial infil tration, following a 6-hour pretreatment with α-throm bin.21 The mechanistic relationship between the rapid and these delayed effects of thrombin on PMNL-endothelial interactions is not yet clear. It is known that cytokines such as interleukin-1 (IL1α) and tumor necrosis factor (TNFα) induce de novo synthesis, by vascular endothelium, of an endothelial leukocyte adhesion molecule (ELAM-1) and up-regulation of the expression of intercellular adhesion mole cule-1 (ICAM-1).26,27 Both of these molecules contrib ute not only to PMNL adhesion, but also to PMNL transendothelial migration in vitro. 28,29 These proinflam matory cytokines also stimulate endothelial cell surface expression of tissue procoagulant or thromboplastin30,31 and down-regulate anticoagulant factors such as throm bomodulin.32 As a result, endothelium involved in in flammation likely promotes blood coagulation. Because of the increasing potential for interactions between blood coagulation and inflammation, the goal of our investiga tions has been to determine the effect of α-thrombin on PMNL interaction with cytokine-activated endothelium, as is likely to occur at sites of inflammation.
MATERIALS AND METHODS Endothelial cells from human umbilical veins (HUVE) were harvested and cultured by the method of Jaffe et al 33 using modifications previously described.34 At confluence, HUVE were trypsinized and isolated cells were grown for 6 days at 37°C to form a tight monolayer on gelatin/fibronectin-coated polycarbonate filters (3 |xm pores) attached to culture plate inserts (Costar, Cam bridge, MA). Using this system, PMNL adherence and transendothelial migration, utilizing 51Cr-labeled human PMNL, can be separately measured, as we previously reported.35 Monolayer permeability was measured using 125 I-albumin. For assays, HUVE monolayer filters were stimu lated for 3 hours from the basal direction (lower chamber) with IL-1α or TNFα in 0.6 ml of RPMI-1640 10% fetal calf serum (FCS). This stimulation was followed by a media change and to the lower chamber 0.6 ml of RPMI1640 0.5% human serum albumin (HSA)-N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) (10
mM) was added. Purified 51Cr-labeled human PMNL (2.5 x 105/0.1 ml) in RPMI-1640-HSA-HEPES contain ing 3-5 x 104 cpm 125I-HSA was added to the chamber above the monolayer/filter unit. At this point, α-throm bin was added, usually to the upper chamber, unless otherwise indicated, and the chemotactic factor f-norLeu-Leu-Phe (FNLP), when used, was added to the chamber below the monolayer. The PMNL and HUVE were incubated at 37°C for 15 to 90 minutes, migration was stopped by washing the surface above the monolayer to remove nonadherent PMNL, and the undersurface of the filter was rinsed and collected into the lower compart ment. The percent of applied 51Cr-PMNL remaining ad herent to the monolayer, the percent that migrated into the lower chamber beneath the monolayer/filter unit, and the percent of 125I-HSA added to the upper chamber that diffused across the monolayer into the lower chamber were determined by a gamma spectrometer. In vivo studies were carried out on a rabbit dermal inflammation model. New Zealand White rabbits of ei ther sex weighing 3 to 4 kg were shaved and 40 to 50 skin sites were designated for intradermal injection. The quantitation of leukocyte accumulation in dermal inflam matory reactions was performed by isolation of rabbit blood leukocytes, labeling them with 51Cr and intrave nous infusion according to previously published meth ods. 36,37 Inflammatory stimuli, including IL-1α, TNFα, NAP-1 (IL-8), zymosan-activated serum (ZAS, contain ing C5a desArg ), 36,38 or control 0.1% HSA phosphatebuffered saline (PBS) were injected intradermally im mediately following intravenous infusion of labeled leu kocytes. Sixty minutes later, at the peak of PMNL migra tion, α-thrombin was injected intradermally into the same and new sites together with intravenous injection of 125 I-HSA. Then, 30 minutes later, after a total of 90 minutes, the animal was sacrificed, the skin removed, and the lesions punched out. The accumulation of 51Cr (on leukocytes) and 125I (on albumin) in the tissue was determined by a gamma spectrometer. The 51Cr radioiso tope content was converted to the number of PMNLs (x 106) accumulated in the lesions during the reaction as previously reported,36 and the 125I content indicating plasma protein leakage was converted to microliters of plasma per site, based on plasma 125I content at sacrifice, as reviewed previously.37 An enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of ELAM-1 and ICAM-1 on the endothelial cell surface, as previously described.35 Briefly, HUVE was grown to confluence in microwells and then stimulated with or without IL-1α for 3 hours. The monolayers were rinsed once with RPMI, and subsequently stimulated with α-thrombin for 10 min utes. Following another rinse with RPMI-1640, mono clonal antibodies H4/18 (anti-ELAM-1), RR 1/1 (anti-
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ICAM-1), and 3H11B9 (antipertussis toxin as negative control) at 1:1000 of ascites were applied in RPMI-1640, 5% FCS with 0.1% azide for 60 minutes at 37°C. These antibodies were removed, the monolayer washed, and then incubated with a 1:3000 dilution of peroxidase con jugated goat antimouse immunoglobulin G (IgG) in RPMI-1640, 5% FCS for 60 minutes at 37°C. Unbound conjugate was removed, and the enzyme substrate, O-phenylenediamine (0.34 mg/ml) in a pH 5 citratephosphate buffer, was then added. Color development was stopped with 2 M sulfuric acid and the optical den sity of each well read at 492 nm in a Biotek ELISA plate reader.
RESULTS Effect of Thrombin on Polymorphonuclear Leukocyte Migration in Vitro Previous studies have shown that α-thrombin can elicit two temporally different forms of PMNL-endothelial interactions.19,21 In our study, α-thrombin (0.1 U/ml) induced rapid, transient, but relatively weak (3.4% ± 1.4 versus control of 0.3 ± 0.1%) adherence of added PMNLs during 15 to 30 minutes of PMNL incuba tion with endothelium. In contrast, IL-1α (0.05 ng/ml) induced adherence of 21.2% ± 2.0 of the PMNL (not shown). Figure 1 shows the time course of PMNL migra tion through unstimulated endothelial monolayers and monolayers treated with α-thrombin (0.1 U/ml) added to the upper chamber (apical surface) at the time of PMNL addition. As shown in Figure 1, α-thrombin stimulation of endothelium was capable of inducing transendothelial
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migration of 13.2% ± 0.62 of the PMNL by 15 minutes, which plateaued between 30 and 90 minutes. A mean PMNL migration of 8.4% ± 0.92 at a 30-minute incuba tion time was seen in 15 other experiments in which α-thrombin (0.1 U/ml) was used to stimulate the endo thelium. This migration did not increase significantly at longer incubation times, indicating the thrombin effect was short-lived for PMNL migration. α-Thrombin-induced PMNL migration was relatively low when com pared with that observed across IL-1α stimulated endo thelium, in which case PMNL migration continued to increase with incubation up to 90 minutes, as shown in Figure 1 and reported previously.35 Very few PMNLs migrated through unstimulated endothelium (less than 5%) at any time. Consistent with observations by other workers, α-thrombin exerted its migration effects via a direct ac tion on the endothelium,21'39 because in the assay system used, α-thrombin was added to the endothelium for 10 minutes, the monolayer washed, and PMNLs subse quently added, so α-thrombin was not available to inter act with the PMNLs. There was no significant difference in the response whether the endothelium was stimulated via the apical surface (thrombin added to upper chamber above monolayer) or basal surface (lower chamber below monolayer; not shown). In this system, α-thrombin did not have a direct PMNL chemotactic effect or induce PMNL migration across bare membranes in the absence of endothelium (not shown). Using the described system, we have previously shown that pretreatment of endothelial monolayers with IL-1α for 2 to 4 hours is optimal for activation of the endothelium for PMNL adherence and migration.35 In view of evidence that α-thrombin shares with IL-1α the
FIG. 1. Kinetics of the enhancement by α-thrombin of interleukin-1a (IL1α)-induced polymorphonuclear leu kocyte (PMNL) transendothelial mi gration. Human umbilical vein endothelial monolayers were unstim ulated, prestimulated for 3 hours with 0.05 ng/ml IL-1α, or stimulated 10 min utes with 0.1 U/ml α-thrombin. PMNLs were added and incubated to allow mi gration for varying periods of time. Values are the percent of added PMNLs that migrated across the monolayer/filter unit. Means ± SD of triplicate determinations are shown. Similar results were obtained in four other time-course experiments. An as terisk denotes significant (p < 0.05) en hancement over additive values.
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ability to activate endothelial cells, we studied the effect of α-thrombin stimulation on endothelium prestimulated with IL-1α, to investigate whether α-thrombin may mod ulate this type of PMNL-endothelial interaction. Figure 1 shows that α-thrombin was able to alter the kinetics of the IL-1 induced migration by increasing, in a synergistic manner, the initial rate of PMNL transendothelial migra tion. At both 15 and 30 minute time points, the migration was enhanced by 58% over additive values. At the later time points, the migration of PMNLs across IL-1α acti vated endothelium, in the absence of thrombin, caught up to the absolute values seen with thrombin addition. We then examined the effect of α-thrombin on PMNL migration across HUVE monolayers induced by TNFα and the synthetic PMNL chemotactic factor FNLP. TNFα is known to activate endothelium for adhe siveness for PMNL analogous to IL-1α, 40 and this also results in PMNL transendothelial migration. 21,28,29,35 In contrast to IL-1α and TNFα, FNLP (3 x 10 - 8 M) does not directly activate HUVE, and when added to the lower chamber, it induces PMNL-transendothelial migration by a direct, receptor-mediated chemotactic effect on PMNL. Figure 2 shows the results of an experiment in which the endothelium was treated with α-thrombin (0.1 U/ml) af ter prestimulation with IL-1α (0.05 ng/ml) or TNFα (20 U/ml), or when FNLP was added into the lower chamber. At a 30-minute PMNL migration period, α-thrombin en hanced, in a synergistic manner, the PMNL migration induced by IL-1α and FNLP and had an additive effect on TNFα-induced migration. To determine whether α-thrombin enhanced PMNL migration via its proteolytic activity, experiments were carried out in the same fashion using α-thrombins in which the catalytic site was irreversibly inhibited by diisopropylfluorophosphate (DIP) or D-phenylalanyl-L-
prolyl-L-arginine chloromethyl ketone (PPACK) treat ment.41 Results in Figure 3 show that these enzymatically inactive thrombins did not enhance PMNL transendothe lial migration. Thus, although DIP-thrombin, PPACKthrombin, and α-thrombin are known to bind the same receptor on endothelium with similar affinity,42,43 the findings indicate that the enhanced PMNL migration in duced by α-thrombin is dependent on enzymatic activity and not only receptor binding.
Effect of Thrombin on Polymorphonuclear Leukocyte Migration in Vivo The observations already described were further evaluated in an in vivo rabbit dermal inflammation model. Initially, we examined the effect of various doses of α-thrombin on dermal leukocyte accumulation and found that intradermal injection of α-thrombin at a dose of 10 U/site induced PMNL accumulation of 1.5 x 106 PMNL/site by 90 minutes. This PMNL infiltration was consistently greater than control (0.1% HSA in PBS) values of 0.2 x 106 PMNL/site, but much weaker than responses to IL-1α (2 ng/site) which reached 16 x 106 PMNL/site. We then examined the effect of α-thrombin on the PMNL accumulation induced by a dose range of IL-1α (0.06 to 2 ng/site) injected intradermally. One hour after the intradermal injection of IL-1α (and after the intrave nous infusion of 51Cr-labeled leukocytes), these sites were reinjected intradermally with 10 U/site of α-throm bin or HSA-PBS control. As shown in Figure 4, in vivo findings corroborated the in vitro results. A significant enhancement of PMNL accumulation by α-thrombin oc curred over a wide dose range of IL-1α, demonstrating that this enhancement is not dependent on the IL-1α
FIG. 2. The effect of α-thrombin (0.1 U/ml) on polymorphonuclear leuko cyte (PMNL) transendothelial migra tion in vitro induced by interleukin-1α (IL-1a) (0.05 ng/ml), tumor necrosis factor-alpha (TNFα) (20 U/ml) and FNLP (3 x 10 -8 M). Human umbilical vein endothelial cells (HUVE) were pretreated for 3 hours with IL-1α or TNFα prior to the addition of thrombin, as in Figure 1. FNLP was added to the lower chamber without pretreatment of HUVE at time of thrombin and PMNL addition. Values are mean ± SD of triplicate determinations and are representative of five experiments. An asterisk denotes significant (p < 0.001) enhancement over additive val ues.
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α-thrombin, resulted in synergistically increased PMNL accumulation (190 to 260%), that is, greater than the additive sum of each response alone, which is expressed as the 100% value. In contrast, no accumulation greater than the 100% additive value was observed with NAP-1 and ZAS, the chemotactic agents with α-thrombin com binations. Our findings thus suggest that α-thrombin's effect on the rate of PMNL transmigration induced by IL-1α observed in the in vitro assay is relevant to the dynamics of PMNL adhesion to and migration across microvessels in vivo.
FIG. 3. Effect of enzymatically inactive thrombins on interleukin-1α (IL-1α)-induced polymorphonuclear leukocytes (PMNL) migration. Human umbilical vein endothelial (HUVE) monolayers were stimulated with 33 ng/ml α-thrombin (0.1 U/ml) or 330 ng/ml of active site inhibited thrombins, diisopropylfluorophosphate (DIP) or D-phenylalanyl-L-prolyl-Larginine chloromethylketone (PPACK), 10 minutes prior to PMNL addition. The effect of these thrombins on PMNL re sponses on HUVE prestimulated for 3 hours with 0.05 ng/ml IL-1 is also shown. Data are presented as mean ± SD of tripli cates, representative of three experiments. PMNL migration was for 30 minutes.
dose. Histologic examination of the lesions confirmed these quantitative differences and that the leukocytic in filtrate was more than 90% PMNLs with the PMNLs in the extravascular connective tissue (not shown). There was no sign of thrombosis with the doses of α-thrombin used. α-Thrombin's capacity to affect PMNL accumula tion induced by other agents known to promote PMNL migration was then studied. TNFα (1000 U/ml), another cytokine known to activate endothelial cells, was used in the same manner as IL-1α. NAP-1 (IL-8; 2 µg/site), a newly characterized peptide cytokine known to be highly chemotactic for PMNLs44 was given intradermally 30 minutes prior to the injection of α-thrombin. Rabbit ZAS, the active component of which is known to be C5a desArg , 38 also a PMNL chemotactic factor, was in jected in a 1:50 dilution 60 minutes prior to α-thrombin. Results in Figure 5 show that combinations of IL-1α followed by α-thrombin, and TNFα followed by
Leukocyte adherence to and migration across an endothelial barrier involves interaction between PMNL surface molecules and ligands expressed on the surface of the endothelium, such as ELAM-1 and ICAM-1. 26-29,45 To evaluate the relative involvement of these two ligands in the enhanced migration induced by α-thrombin across IL-1α and TNFα stimulated endothelium, an ELISA was used to measure the expression of these molecules on the endothelial cell surface. As shown in Table 1, there was no significant change in the expression of either of these ligands in response to stimulation with α-thrombin. Thus, the enhanced migration seen in response to α-thrombin stimulation of IL-1α prestimulated endothe lium does not appear to be due to an up-regulation of these ligands on the endothelial cell surface. Receptor-mediated agonists, such as α-thrombin, have been shown to induce rapid mobilization of intracel lular stores of the adhesion glycoprotein GMP-140 to the endothelial surface.22 Structurally similar to ELAM-1, 46 GMP-140 appears to serve as a receptor for PMNL glyco proteins, especially LECAM-1 (Mel 14).23 An additional mechanism, recently recognized to contribute to endo thelial cell-PMNL interactions in response to α-throm bin, is PAF expression on the endothelial cell membrane, likely via PAF receptors on the PMNL. 19,25 To evaluate the contribution of GMP-140 in our observations, eight experiments were performed with blocking anti-GMP140 monoclonal antibody Gl and the nonblocking mAb S12 (as a negative control) to evaluate the effect on the enhancement by α-thrombin of PMNL migration across IL-1α-stimulated endothelium. The HUVE was stimu lated with α-thrombin for 10 minutes in the presence of these monoclonal antibodies before PMNL were added. To evaluate the role of HUVE-generated PAF, four ex periments were also carried out in which PMNL were pretreated with various doses (0.5 to 50 µg/ml) of the PAF antagonists, WEB-2086 or CV3988, before their addition to the HUVE. No consistent inhibition of the
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FIG. 4. α-Thrombin enhancement of interleukin-1α (IL-1α)-induced dermal polymorphonuclear leukocyte (PMNL) accumulation in rabbits. 51Cr-labeled leukocytes were injected intravenously and skin sites received IL-1α in a dose range of 0.06-2 ng/site or 0.1% human serum albumin-phosphate buffered saline (HSA-PBS) control injected intradermally (0.2 ml). Sixty minutes later, the same sites were reinjected intradermally with 0.1 ml of 10 U/site α-thrombin or 0.1% HSA-PBS control. At the time of sacrifice, all lesions were 90 minutes of age. Control (HSA-PBS) injection alone did not induce significant PMNL accumulation (0.2 x 106 PMNL/site). The left hand bar in each pair gives the proportional breakdown of the sum of the thrombin alone and IL-1 alone responses. Values are mean ± SD of triplicate sites, representative of five experiments. An asterisk denotes significant (p < 0.05) enhancement over additive values. All doses and combinations were performed in the same animal.
thrombin enhancement of PMNL migration was ob served using either the anti-GMP-140 monoclonal anti bodies or the PAF antagonists (data not shown). It ap pears that these mechanisms, at least individually, are not responsible for the α-thrombin enhancement of PMNL migration across IL-1 -activated HUVE and complemen tary responses, perhaps with other ligand-receptor sys tems, may be involved.
SUMMARY AND CONCLUSIONS Interactions of leukocytes with the vascular endo thelium is fundamental to many aspects of immune and inflammatory responses. In this report, the ability of α-thrombin, a key enzyme in the coagulation cascade, to alter endothelial cell adhesiveness for PMNL and to stim ulate their transmigration was examined. Both our in vitro and in vivo findings suggest that α-thrombin by itself activates relatively weak endothelial adhesion, as reported by others, and is a weak stimulus for PMNL migration. However, more significantly, our findings have shown that α-thrombin synergistically increases the PMNL transmigration in vitro and PMNL accumulation in vivo induced by IL-1α. This suggests that the en hanced rate of PMNL transmigration observed in vitro with α-thrombin plus IL-1α is relevant to the dynamics of
PMNL adhesion and migration across microvessels in vivo. The results demonstrate an accessory role for α-thrombin-dependent mechanisms, perhaps to stabilize or amplify the adhesion mechanisms induced by IL-1α or TNFα, leading to immobilization of increased numbers of PMNL in the microvessels under shear stress condi tions, and thus result in enhanced PMNL transendothelial migration and tissue infiltration. In recent years, potential proinflammatory actions of α-thrombin have been recognized, especially with re gard to vascular endothelial permeability and leukocyteendothelial interactions. With the growing interest in the potential interactions between inflammation and the co agulation cascade, our results may be relevant to a wide variety of inflammatory reactions in which a procoagulant environment can develop, potentially leading to α-thrombin generation at the endothelial cell surface. Thus, further studies elucidating the in vivo contribution of thrombin in a variety of inflammatory reactions appear warranted. Studies utilizing PAF antagonists in vivo as well as the specific thrombin inhibitor hirudin, to further determine thrombin's role in inflammatory processes, are under way. Acknowledgments: The authors are grateful for gifts of monoclonal antibodies H4/18 from Dr. M. Gimbrone (Harvard Medical School, Boston, MA), RR 1/1 from Dr. T. Springer
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TABLE 1. Effect of α-Thrombin and lnterleukin-1α on Endothelial Cell Expression of ELAM-1 or ICAM-1
HUVE Treatment*
ELAM-1
ICAM-1
Unstimulated
