Departments of 1 Medicine, 2 Laboratory Medicine, and 3 Dermatology, Kumamoto University School of Medicine, Kumamoto 860; and 4 Institute for Life Science Research Laboratory, Asahi Chemical Industry, Shizuoka 410-23, Japan
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ABSTRACT |
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We have previously demonstrated that recombinant human soluble (rhs) thrombomodulin (TM) inhibits the endotoxin (ET)-induced increase in pulmonary vascular permeability by inhibiting leukocyte activation. In the present study, we examined whether rhs-TM could inhibit the ET-induced increase in pulmonary vascular permeability in rats by activating protein C. rhs-TM did not inhibit ET-induced increases in pulmonary vascular permeability when its protein C activation ability was selectively inhibited by a monoclonal antibody (MAb) against rhs-TM (MAb R5G12). Histological examination revealed that neutrophil infiltration in lung tissues after ET administration was significantly reduced by rhs-TM, but infiltration was not reduced by MAb R5G12-pretreated rhs-TM. ET-induced intravascular coagulation was prevented by rhs-TM and by MAb R5G12-pretreated rhs-TM. However, ET-induced coagulation was not prevented by rhs-TM that had been treated with MAb F2H5, which cannot bind thrombin or activate protein C. These observations strongly suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes through thrombin binding and the subsequent protein C activation and may prevent ET-induced intravascular coagulation through thrombin binding.
acute respiratory distress syndrome; disseminated intravascular coagulation; leukocyte activation; cytokines; endothelial cell injury ; recombinant human soluble thrombomodulin; endotoxin
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INTRODUCTION |
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ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is one of the critical pathological events in sepsis that is associated with a mortality rate of >50% (4, 21). Cytokines and other inflammatory mediators derived from activated leukocytes have been implicated in the pathogenesis of ARDS (22, 29).
Thrombomodulin (TM) is an endothelial cell membrane glycoprotein that forms a high affinity complex with thrombin (20). TM plays an important role in the downregulation of coagulation (8). Thrombin bound to TM does not show procoagulant activity, such as conversion of fibrinogen into fibrin and the activation of factor V and VIII, but it can activate protein C to form activated protein C (APC) in plasma (8). APC thus formed exerts anticoagulant effects by inactivating factors Va and VIIIa, thereby regulating the coagulation cascade (23, 28).
TM also plays an important role in downregulation of lung injury. Recently, we demonstrated that recombinant human soluble (rhs) TM prevented endotoxin (ET)-induced pulmonary vascular injury by inhibiting leukocyte activation as well as the coagulation abnormalities in rats (24). Because APC has been implicated in the regulation of inflammatory reactions involving cytokines or activated leukocytes (7) and because we have previously demonstrated that APC prevented ET-induced pulmonary vascular injury by inhibiting leukocyte activation in rats (16), rhs-TM might inhibit the ET-induced pulmonary vascular injury by APC generation. However, because thrombin plays an important role in the pathophysiology of ARDS (10), it is possible that rhs-TM/thrombin binding may also contribute to the prevention of ET-induced pulmonary vascular injury in rats.
The present study was undertaken to investigate whether protein C activation by rhs-TM is responsible for prevention of ET-induced pulmonary vascular injury, as measured by inhibition of ET-induced leukocyte activation.
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MATERIALS AND METHODS |
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Materials. rhs-TM was kindly provided by Asahi Chemical Industry (Tokyo, Japan); the method used for preparation of rhs-TM has been described previously (9). Purified rhs-TM migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9). This form of rhs-TM has plasma half-lives of 0.2 and 7 h in rats (2). Bovine serum albumin (BSA) was obtained from Sigma Chemical (St. Louis, MO), ET (lipopolysaccharide, Escherichia coli, serotype 055:B5) was from Difco (Detroit, MI), and Bolton-Hunter reagent and 51Cr were from Amersham International (Buckinghamshire, UK). A chromogenic substrate, S-2366 (Glu-Pro-Arg-p-nitroanilide), was obtained from Chromogenix (Stockholm, Sweden). All reagents used were of analytical grade.
Preparation of monoclonal antibodies against rhs-TM. Anti-rhs-TM monoclonal antibodies (MAbs) MF2, R5G12, and F2H5 were produced by the hybridoma technique in mice using rhs-TM as the antigen (12). In brief, the hybridoma supernatants obtained from BALB/c mice immunized with rhs-TM were assayed for antibody production. The selected hybridoma clones were expanded by intraperitoneal injection of hybridomas into BALB/c mice pretreated with pristane (Wako Chemical, Osaka, Japan). The ascites fluid was collected 1-2 wk after the injection. The immunoglobulin G antibodies were affinity purified from mouse ascites under sterile conditions on protein A Sepharose CL-4B.
Measurement of thrombin binding activity and protein C activation capacity of rhs-TM. Effects of various MAbs on thrombin binding to rhs-TM and protein C activation capacity of rhs-TM were determined according to a previously described method (1). Because fibrinogen clotting activity of thrombin is inhibited by rhs-TM/thrombin binding, thrombin-mediated fibrinogen clotting time was measured in the presence of rhs-TM and various MAbs to evaluate the thrombin binding activity by rhs-TM. Clotting time was measured at 37°C in a coagulometer (KC4A; Amelung, Lieme, Germany). rhs-TM (50 nM) was preincubated at 37°C for 2 min with 50 nM of MAb in 50 µl of a 25 mM tris(hydroxymethyl)aminomethane (Tris) · HCl buffer (pH 7.5) containing 0.15 M NaCl and 0.02% of Lubrol PX. After 50 µl of 3 mg/ml bovine fibrinogen in 25 mM Tris · HCl buffer (pH 7.5) containing 0.15 M NaCl were added to the preparation, 50 µl of 50 nM thrombin were added to initiate clotting.
Protein C activation by rhs-TM was evaluated as follows. rhs-TM (1 mM) was pretreated with an equimolar amount of various MAbs in 20 mM Tris · HCl (pH 7.5) containing 0.15 M NaCl, 5 mM CaCl2, and 0.5% BSA. After an incubation for 30 min at 37°C, thrombin and protein C were added at final concentrations of 1 mM and 0.15 µM, respectively. The reaction mixture was incubated at 37°C for 1 min. The reaction was terminated by the addition of 0.5 mg/ml argatroban. APC generation was measured spectrophotometrically (DU-64; Beckman, Irvine, CA) as the increase in absorbance at 405 nm after the addition of 0.5 mM of S-2366.Treatment of rhs-TM with MAbs MF2, R5G12, and F2H5. rhs-TM was incubated at 37°C for 30 min with an equimolar concentration of MAb MF2, R5G12, or F2H5. Aliquots of the solution were assayed for protein C activation capacity and fibrinogen clotting activity, as described above, to determine the TM activity.
Animal model of ET-induced acute pulmonary vascular injury.
The study protocol was approved by the Kumamoto University Animal Care
and Use Committee, and the care and handling of the animals were in
accordance with National Institutes of Health guidelines. Adult
pathogen-free male Wistar rats (body mass 180-220 g; Nihon SLC,
Hamamatsu, Japan) received an intravenous infusion of
125I-labeled BSA [specific
activity, 3.0 × 1014
counts · min1
(cpm) · mol
1]
prepared with Bolton-Hunter reagent (2.0 × 105 cpm/kg body wt) 5 min before
an intravenous bolus dose of 5 mg/kg ET was injected into the tail vein
as described previously (16). rhs-TM (1 mg/kg) treated with or without
MAbs against rhs-TM was administered intravenously 30 min before
injection of ET. Control animals received saline only. Animals were
anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital
sodium and were exsanguinated via the abdominal aorta 6 h after
administration of ET. Blood samples were collected in tubes containing
a 0.1 volume of 3.8% sodium citrate. Blood was centrifuged at 2,000 g for 10 min. The lung vasculature was
perfused through the right cardiac ventricle with 10 ml of saline. The
lungs were then removed and weighed, and the amount of radioactivity
remaining within the tissue was measured with a gamma scintillation
counter (model 5130; Packard Instrument, Downers Grove, IL). Pulmonary
vascular damage induced by ET was evaluated by increased vascular
permeability as expressed by a permeability index. The permeability
index is defined as the distribution ratio of
125I-BSA between lung and blood,
that is, the ratio of the amount of radioactivity present within lung
tissue to the amount of radioactivity present in 1 ml of blood obtained
at the time of death, as described previously (16). The change in
pulmonary vascular permeability evaluated by this method was well
correlated with the change in wet-to-dry weight ratio of rat lungs
after ET challenge (27).
Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed as described previously (24). In brief, after intraperitoneal administration of pentobarbital sodium, an incision was made in the anterior neck, and a catheter was secured in the trachea with a surgical suture. BAL was performed with 5 ml of saline. Radioactivity in the BAL fluid was measured with a gamma scintillation counter.
Measurement of lung myeloperoxidase activity. Ninety minutes after ET administration, 10 ml of saline were perfused through the lung vasculature into the right cardiac ventricle. The lungs were then removed, and accumulation of leukocytes was assessed by measuring myeloperoxidase (MPO) activity in rat lungs according to a previously described method (16). In brief, lung samples were homogenized using 6 ml of homogenizing buffer containing 0.1 M phosphate buffer (pH 6.0) and 1% hexadecyltrimethylammonium bromide. The homogenate was then sonicated and was centrifuged at 4,500 g for 30 min at 4°C. The supernatant was measured for MPO activity as follows. The 0.1-ml test sample was mixed with 0.6 ml of 0.05 M phosphate buffer containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% H2O2 (pH 6.0). The change in absorbance at 460 nm was measured over 1 min at 25°C in a spectrophotometer (DU-54; Beckman). One unit of enzyme activity was defined as the amount of MPO present that causes a change in absorbance of 1.0 per minute at 460 nm and was expressed as units per gram of lung weight.
Histopathological studies of the lungs. Histopathological examination of the lungs was performed 6 h after administration of 5 mg/kg ET. Samples were fixed with 10% Formalin, embedded in paraffin, sectioned into 6-µm pieces, and stained with hematoxylin and eosin. Samples were analyzed by a pathologist blinded to the animal's group assignment. Ten randomly selected fields per slide were read under oil at ×1,000 magnification by an observer who did not know the animal's group assignment. Fields containing large vessels or bronchi were excluded. The number of polymorphonuclear neutrophils per field was counted and normalized to alveoli per field to control for inflation of the lung.
Assays. The plasma concentration of fibrinogen was determined 6 h after ET administration as the amount of coagulable protein, according to the method of Clauss (6). Fibrin and fibrinogen degradation products (E) [FDP(E)] were measured 6 h after ET administration in rat serum samples with the latex agglutination assay, as previously described (16).
Statistical analysis. Data are presented means ± SD. Data were analyzed by analysis of variance and Scheffé's post hoc test. P < 0.05 was accepted as statistically significant.
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RESULTS |
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Effects of MAbs on thrombin binding activity and protein C activation capacity of rhs-TM. MAb F2H5 almost completely inhibited thrombin binding to rhs-TM, but MAbs R5G12 and MF2 did not inhibit thrombin binding to rhs-TM (Fig. 1). The protein C activation capacity of rhs-TM was almost completely inhibited by MAb F2H5 and was inhibited by MAb R5G12 by ~66% but was not inhibited by MAb MF2 (Fig. 2). Thus MAb F2H5 inhibits thrombin binding and subsequent protein C activation by rhs-TM, and MAb R5G12 inhibits protein C activation by rhs-TM. MAb MF2 does not affect thrombin binding to rhs-TM or subsequent protein C activation. These activity experiments were repeated using the solutions that were administered to rats for testing (data not shown). These solutions comprised rhs-TM, various MAbs, and saline.
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Effects of MAb-treated rhs-TM on ET-induced pulmonary accumulation of leukocytes, increase in pulmonary vascular permeability, and intravascular coagulation in rats. In this animal model of septicemia, pulmonary accumulation of leukocytes, as evaluated by determining lung MPO activity, increases with time and peaks at 90 min after ET administration. Pulmonary vascular permeability to 125I-BSA increases with time after administration of ET and peaks at 6 h of ET administration (16). Intravenous administration of 1 mg/kg rhs-TM significantly inhibits (1) the increase in pulmonary accumulation of leukocytes 90 min after ET administration and (2) the increase in pulmonary vascular permeability 6 h after ET administration (16). rhs-TM (1 mg/kg) also attenuates intravascular coagulation induced by ET at 6 h (24). There is no significant increase in the ratio of 125I-BSA concentration in the BAL fluid to that in blood 6 h after administration of ET.
To determine whether rhs-TM might inhibit the increase in pulmonary vascular permeability by binding thrombin or by activation of protein C, we examined the effect of MAb-treated rhs-TM on the ET-induced pulmonary accumulation of leukocytes and the subsequent increase in pulmonary vascular permeability. Pretreatment of rhs-TM with MAbs F2H5 or R5G12 did not prevent the ET-induced pulmonary accumulation of leukocytes or the increase in pulmonary vascular permeability observed at 90 min and 6 h after ET administration, respectively (Fig. 3, A and B). Pretreatment of rhs-TM with the MF2 MAb prevented both the pulmonary accumulation of leukocytes and the increase in pulmonary vascular permeability (Fig. 3, A and B). As stated earlier, MAb MF2-treated rhs-TM can activate protein C and can bind thrombin.
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Effects of rhs-TM and rhs-TM pretreated with MAb on changes in the pulmonary histological findings induced by ET. Microscopic observation of lung tissue 6 h after administration of ET revealed edematous changes and infiltration of neutrophils in the interstitial space (Fig. 4B). These changes were not present in lung tissue from animals treated with saline alone (Fig. 4A). Administration of rhs-TM markedly reduced interstitial edema and neutrophilic infiltration (Fig. 4C). Administration of MAb MF2-treated rhs-TM also reduced interstitial edema and neutrophilic infiltration (Fig. 4F). However, administration of MAb R5G12-treated rhs-TM or F2H5-treated rhs-TM did not reduce ET-induced histological changes in the lung (Fig. 4, D and E).
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DISCUSSION |
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In the present study, the anti-inflammatory activity of rhs-TM was
correlated with the protein C activating ability of rhs-TM. In
particular, rhs-TM did not inhibit the ET-induced increase in pulmonary
vascular permeability when the protein C activation ability of rhs-TM
was selectively inhibited by pretreatment of rhs-TM with MAb R5G12.
This suggests that inhibition of the ET-induced increase in pulmonary
vascular permeability by rhs-TM may be dependent on the ability of
rhs-TM to activate protein C but is not dependent on its ability to
bind thrombin, although rhs-TM pretreated with MAb F2H5 failed to
prevent the ET-induced increase in pulmonary vascular permeability. The
observations in the present study are consistent with our previous
report demonstrating that APC prevents the ET-induced increase in
pulmonary vascular permeability (16). In addition, we have recently
demonstrated that APC may prevent the ET-induced increase in pulmonary
vascular permeability by inhibiting tumor necrosis factor- (TNF-
)
production by monocytes in rats (17). TNF-
activates neutrophils to
release the various inflammatory mediators capable of damaging
endothelial cells (1, 13). Thus rhs-TM may inhibit the ET-induced
increase in pulmonary vascular permeability through generation of APC,
which can inhibit leukocyte activation.
The fact that R5G12-treated rhs-TM did not inhibit ET-induced pulmonary vascular permeability suggests that procoagulant activity of thrombin may not be involved in the pathological process leading to this permeability in the animal septicemia model used in this study. However, coagulation abnormalities have been considered to be an important predisposing factor for ARDS (4, 15). The fibrin and fibrin degradation products present in the alveolar spaces have been shown to be chemoattractants for leukocytes that play an important role in the pathogenesis of ARDS (3, 11). Because we did not observe fibrin deposition in the alveolar spaces and because the amount of 125I-BSA intravenously injected was not increased in BAL in rats given ET, ET-induced lung injury might be limited to the endothelial cells in the animal model of septicemia used in the present study. This may explain why inhibition of intravascular coagulation did not attenuate the ET-induced increase in pulmonary vascular permeability in the present study. However, prevention of intravascular coagulation by rhs-TM, which is mediated mainly by binding thrombin, may contribute to the attenuation of ARDS in a clinical setting.
One may speculate that the pretreatment of rhs-TM with any MAb adversely affects the ability of rhs-TM to prevent the ET-induced increase in pulmonary vascular permeability. However, this seems unlikely because treatment of rhs-TM with MAb MF2, which affects neither thrombin binding nor protein C activation by rhs-TM, did not affect the ability of rhs-TM to prevent the ET-induced increase in pulmonary vascular permeability.
We have previously reported that antithrombin III (ATIII) prevents ET-induced pulmonary vascular injury by inhibiting leukocyte activation by stimulating endothelial release of prostacyclin (25). For the stimulation of prostacyclin release from endothelial cells, interaction of ATIII with glycosaminoglycans on endothelial cells is important (26). These observations, together with the present findings, suggest that both the ATIII-glycosaminoglycan system and the TM-protein C system, two major important anticoagulant mechanisms operating on the endothelial cell surface, may regulate the leukocyte activation as well as the coagulation system. Thus these two physiological anticoagulants may be useful for prevention of intravascular coagulation and ARDS in patients with septicemia in which leukocytes play an important role (18, 19).
Whereas the present studies were carried out with soluble TM, this protein is an endothelial cell surface protein. TM is highly enriched in the pulmonary vascular lining (14). Because TM binds thrombin to activate protein C in the pulmonary microcirculation, it is possible that TM may play an important role not only in modulation of coagulation but also in prevention of pulmonary endothelial cell injury by regulation of the activation of leukocytes. Further studies should be done to evaluate such a novel role of endothelial TM in the pulmonary microcirculation.
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FOOTNOTES |
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Address for reprint requests: K. Okajima, Dept. of Laboratory Medicine, Kumamoto Univ. School of Medicine, Honjo 1-1-1, Kumamoto 860, Japan.
Received 14 February 1997; accepted in final form 8 July 1997.
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