Thrombin-Activatable Fibrinolysis Inhibitor Protects against Acute Lung Injury by Inhibiting the Complement System Masahiro Naito1,4, Osamu Taguchi1, Tetsu Kobayashi1, Takehiro Takagi1, Corina N. D’Alessandro-Gabazza1,2, Yuki Matsushima2, Daniel Boveda-Ruiz2, Paloma Gil-Bernabe2, Takahiro Matsumoto2, Ayshwarya-Lakshmi ChelakkotGovindalayathil2, Masaaki Toda2, Atsushi Yasukawa2, Osamu Hataji4, John Morser5, Yoshiyuki Takei3, and Esteban C. Gabazza2 1 Department of Pulmonary and Critical Care Medicine, 2Department of Immunology, and 3Department of Gastroenterology and Hepatology, Mie University Graduate School of Medicine, Tsu, Mie, Japan; 4Respiratory Center, Matsusaka Municipal Hospital, Matsusaka City, Tonomachi, Japan; and 5Department of Hematology, Stanford School of Medicine, Stanford, California
Acute lung injury (ALI) is a devastating disease with an overall mortality rate of 30 to 40%. The coagulation/fibrinolysis system is implicated in the pathogenesis of ALI. Thrombin-activatable fibronolysis inhibitor (TAFI) is an important component of the fibrinolysis system. Recent studies have shown that the active form of TAFI can also regulate inflammatory responses by its ability to inhibit complement C3a, C5a, and osteopontin. We hypothesized that TAFI might have a protective role in ALI. To demonstrate this hypothesis, the development of ALI was compared between wild-type (WT) and TAFI-deficient mice. ALI was induced by intratracheal instillation of LPS. Control mice were treated with saline. Animals were killed 24 hours after LPS. The number of inflammatory cells and the concentration of total protein and inflammatory cytokines were significantly increased in bronchoalveolar lavage fluid from LPS-treated, TAFI-deficient mice compared with their WT counterparts. Significantly higher concentrations of C5a were found in bronchoalveolar lavage fluid and plasma in LPS-treated TAFI knockout mice compared with WT mice. Pretreatment with inhaled C5a receptor antagonist blocked the detrimental effects of TAFI deficiency to levels found in WT mice. Our results show that TAFI protects against ALI, at least in part, by inhibiting the complement system. Keywords: coagulation; lung injury; inflammation; fibrinolysis
Acute respiratory distress syndrome (ARDS) is a devastating disease with a mortality rate ranging between 24 and 48% depending on the severity of hypoxemia (1–3). The incidence of the disease (Received in original form November 12, 2012 and in final form May 12, 2013) This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Author Contributions: M.N. prepared the mouse models and wrote the first draft of the manuscript. O.T. helped in the design of the experiments and in the interpretation of the data. T.K. collaborated in the preparation of lung injury model and histological examinations. T.T. performed collaborated in the mouse models and immunoassays. C.N.D.-G. prepared the mice, performed several measurements, and interpreted the data. Y.M., D.B.-R., and P.G.-B. performed the preliminary experiments in wild-type mice and cooperated in the immunoassays. T.M., A-L.C.-G., A.Y., and O.H. performed histological staining and analysis of data. M.T. performed mRNA expression study and several biochemical analyses. J.M. and Y.T. cooperated in manuscript writing and in the intellectual interpretation of the data. E.C.G. coordinated all aspects of the study. Correspondence and requests for reprints should be addressed to Esteban C. Gabazza, M.D., Ph.D., Department of immunology, Mie University School of Medicine, Edobashi 2-174, Tsu-city, Mie, Japan 514-8507. E-mail:
[email protected]. mie-u.ac.jp This article has an online supplement, which is accessible from the issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 49, Iss. 4, pp 646–653, Oct 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2012-0454OC on May 30, 2013 Internet address: www.atsjournals.org
CLINICAL RELEVANCE Acute lung injury (ALI)/acute respiratory distress syndrome is a frequent clinical disease with a still-high mortality rate. There is an urgent need of novel therapeutic modalities that can improve the outcome of patients with the disease. In the present study, we demonstrate for the first time that thrombin-activatable fibrinolysis inhibitor is protective against ALI, suggesting its potential application for clinical use.
shows geographical variations; there are 65 to 79 cases/100,000 persons annually in the United States and 7 to 34 cases/100,000 persons annually in Northern Europe (3, 4). The lungs of patients with ARDS are pathologically characterized by an initial exudative phase with increased release of inflammatory cytokines, neutrophil infiltration, and vascular endothelial/alveolar epithelial injury, a proliferative phase with enhanced proliferation of alveolar epithelial cells and extracellular matrix deposition, and a terminal fibrotic phase with collagen deposition and lung functional failure (5, 6). There is no curative therapy for patients with ARDS (3, 6). Several clinical trials with anti-inflammatory agents, including corticosteroids and pulmonary vasodilators, have failed to reduce mortality (3, 6). The present standard therapy includes mechanical ventilation with strategic adjustment to avoid ventilator-associated lung injury and general supportive care to attenuate multiorgan system failure (3). Thus, the development of novel drugs could improve the clinical outcome of the disease. Thrombin-activatable fibrinolysis inhibitor (TAFI; procarboxypeptidase B2, procarboxypeptidase U) is a procarboxypeptidase produced by several cells, including hepatocytes, macrophages, and megakaryocytes. Its active form (TAFIa) induces the inactivation of peptides and proteins by removing the arginine and lysine residues from their carboxy-terminus (7, 8). Activation of TAFI can be induced by the thrombin–thrombomodulin complex, plasmin, or thrombin (8). TAFIa inhibits fibrinolysis by cleaving the lysine residues of degraded fibrin and thus abrogating tissue plasminogen activator–mediated activation of plasminogen to plasmin (9). In addition to its antifibrinolytic activity, TAFIa exerts its antiinflammatory effects by inactivating complement factors C5a and C3a and bradykinin (8, 10). The proof-of-concept regarding the anti-inflammatory activity of TAFIa was given by several in vivo studies performed in animal models of human disease; TAFI deficiency exacerbated airway hyper-responsiveness and allergic bronchial inflammation in a mouse model of bronchial asthma and inflammatory arthritis in a model of autoimmune arthritis (11, 12). However, the role of TAFIa in the pathogenesis of acute lung injury (ALI)/ARDS has not been investigated.
Naito, Taguchi, Kobayashi, et al.: TAFI in ALI
In the present study, we hypothesized that TAFI may play a role in the pathogenesis of ALI by inactivating complement factor C5a. To demonstrate this hypothesis, we compared the development of LPS-induced ALI in wild-type (WT) and TAFI knockout (KO) mice.
MATERIALS AND METHODS LPS was purchased from Sigma-Aldrich (St. Louis, MO). Complement C5a receptor antagonist (C5aRA) was prepared by Biomatik Corporation (Cambridge, ON, Canada) (13, 14).
Animals TAFI-deficient mice have been previously well characterized. The WT and TAFI-deficient mice used in the experiments were C57BL/6 (15). Male mice (8 wk old) weighing 19 to 22 g were used in the experiments; WT mice used as controls were purchased from Nihon SLC and maintained in the animal house of Mie University. All animals were kept in the standard mouse unit on a constant 12-hour light/12-hour dark cycle and in a temperature- and humidity-controlled room and were given ad libitum access to food and water. The Committee of Mie University for Animal Investigation approved the experimental research protocol.
ALI in TAFl-Deficient Mice The animals were categorized into the following groups: WT/LPS (n ¼ 5) and TAFI KO/LPS (n ¼ 5) groups received intratracheal instillation of LPS (150 mg/mouse) dissolved in physiological saline (SAL) (75 ml) under anesthesia (62.5 mg/kg intraperitoneal sodium pentobarbital), and the WT/SAL (n ¼ 5) and TAFI KO/SAL (n ¼ 5) received intratracheal instillation of saline (75 ml) under anesthesia. These experiments were performed independently three times in a similar fashion, but representative results from one individual experiment are shown in the figures. Mice of each group were killed at 24 hours for sampling. In addition to the above groups, in two separate experiments, a group of WT (WT/C5aRA/LPS [n ¼ 4]) and a group of TAFI KO (TAFI KO/ C5aRA/LPS [n ¼ 5]) mice were pretreated with C5aRA (1,000 mg/mouse) by inhalation using a nebulizer (MIPS, Osaka, Japan) 24 and 2 hours before intratracheal instillation of LPS (150 mg/mouse) to evaluate the role of complement activation in TAFI KO–associated ALI. Mice receiving intratracheal saline alone (WT/SAL [n ¼ 3], TAFI KO/SAL [n ¼ 4 z 5]) or intratracheal saline 24 and 2 hours before intratracheal LPS instillation (WT/SAL/LPS [n ¼ 4 or 5], TAFI KO/SAL/LPS [n ¼ 6]) were used as controls. Mice in all the above groups were killed at 24 hours for sampling.
Biochemical Analysis The total concentration of protein in BALF was measured using the dyebinding assay (Bio-Rad Laboratories, Hercules, CA). Concentrations of inflammatory cytokines, including IL-6, IL-1b, and TNF-a, were measured using ELISA kits from BD Biosciences Pharmingen (San Diego, CA) following the manufacturer’s instructions. Mouse-specific thrombin– antithrombin (TAT) complexes and D-dimers were measured using enzyme immunoassay kits from Cusabio Biotech (Wuhan, China) following the manufacturer’s instructions. The concentration of plasminogen activator inhibitor (PAI)-1 was measured by enzyme immunoassay using murine anti–PAI-1 (Molecular Innovations, Southfield, MI) antibody with biotin-labeled antimouse antibody.
Statistical Analysis Data were expressed as mean 6 SEM. The statistical difference between variables was calculated by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. The distribution of the data was calculated using the Kolmogorov-Smirnov test. Statistical analyses were performed using the StatView 4.5 package for Macintosh (Abacus Concepts, Berkeley, CA). A P value , 0.05 was considered to be significant.
RESULTS Dose-Dependent Effect of LPS Instillation in the Lung of WT Mice
Previous reports of low-dose LPS instillation into the lungs of mice had failed to detect complement system activation (16,
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17). We investigated if higher doses of LPS could trigger complement activation. Mice were instilled with saline (WT/SAL) or with 100 (WT/LPS 100 mg), 150 (WT/LPS 150 mg), or 200 mg (WT/LPS 200 mg) of LPS, and the number of total cells and neutrophils in BALF and the lung tissue concentrations of C5a and Factor D were evaluated. Six hours after LPS instillation, the total number of BALF cells in the WT/150 mg group and the neutrophil count in WT/150 mg and WT/200 mg groups were significantly increased compared with the WT/SAL group. Twenty-four hours after LPS instillation, the total number of cells in the WT/LPS 100 mg, WT/ LPS 150 mg, and WT/LPS 200 mg groups and the neutrophil count in the WT/LPS 150 mg and WT/LPS 200 mg groups were significantly increased compared with the WT/SAL group (see Figures E1A–E1C in the online supplement). The concentrations of C5a and Factor D were significantly increased in the WT/LPS 150 mg and WT/LPS 200 mg groups compared with the WT/SAL group 6 hours after LPS instillation (Figures E1D and E1E). Similarly, the concentrations of C5a and Factor D were significantly increased in all LPS-treated groups compared with the WT/SAL group 24 after hours LPS instillation (Figures E1F and E1G), showing that high-dose LPS could lead to complement activation. Based on these data, 150 mg/mouse of LPS was selected as the dose for the rest of the investigation. Lung Cell Infiltration by Neutrophils and TAFI Deficiency
Because TAFIa has been shown to inactivate C5a, we investigated if mice that were deficient in C5a would have altered outcomes after instillation of LPS. The total number of cells and neutrophils counted after Giemsa staining of lung sections was increased after treatment with intratracheal LPS (WT/LPS and TAFI KO/LPS) compared with mice receiving intratracheal saline (WT/SAL and TAFI KO/SAL). TAFI was shown to play a role in this model because the number of total cells and neutrophil infiltrating the lung was higher in the TAFI KO/LPS group than in the WT/LPS group (Figures 1A and 1B). Histological evaluation of the number of infiltrating cells in the lungs disclosed similar results; inflammatory cells were increased in the mice treated with LPS compared with saline-treated mice, but they were remarkably increased in the TAFI KO/LPS group compared with the WT/LPS group (Figures E2A and E2B). Lung Inflammation and Coagulation System
To confirm these data, an alternate general marker of lung inflammation—the amount of protein leakage into the alveolar space—was measured. As expected, the concentration of total protein in BALF was significantly increased in the WT/LPS and TAFI KO/LPS groups compared with the WT/SAL and TAFI KO/SAL groups, but it was remarkably increased in the TAFI KO/LPS compared with the WT/LPS group (Figure 2A). TAT was measured as a marker of coagulation system activation. The BALF concentrations of TAT complexes were significantly increased in the WT/LPS group, but not in the TAFI KO/LPS group, compared with the saline control group (Figure 2B). The concentration of TAT in lung tissue was significantly increased in mice treated with LPS (WT/LPS and TAFI KO/ LPS) compared with those treated with saline (WT/SAL and TAFI KO/SAL), but it was not significantly different between the TAFI KO/LPS and WT/LPS groups (Figure 2C). The level of D-dimer, a marker of fibrinolysis activity, was lower in the WT/LPS group compared with the WT/SAL group but did not reach significance; there was no significant difference in the level of D-dimer between the TAFI KO/LPS and TAFI KO/ SAL groups, but a significant difference was found between the TAFI KO/LPS and WT/LPS groups (Figure 2D).
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Figure 1. Intra-alveolar cell infiltration. Bronchoalveolar lavage fluid (BALF) was sampled 24 hours after LPS instillation. BALF was centrifuged by cytospin and stained with Giemsa. (A and B) The total number of BALF cells and the number of neutrophils were significantly increased in LPStreated mice compared with saline-treated mice, but there was also a significant difference in the total cell number and neutrophils between the wild-type (WT)/LPS and the thrombin-activatable fibronolysis inhibitor (TAFI) knockout (KO)/LPS groups (n ¼ 5 mice in each group). The figure shows representative results from one of three independent experiments. Bars indicate the means 6 SEM. Scale bars indicate 50 mm. Statistical analysis by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. *P , 0.05 versus the respective saline groups; xP , 0.05 versus WT/LPS. SAL, saline.
PAI-1 is an inhibitor of plasminogen activators that has a different mechanism than TAFI for inhibition of fibrinolyis and is frequently induced in inflammatory situations (18). The concentration of PAI-1 in plasma, BALF, and lung tissue homogenates was significantly increased in the TAFI KO/LPS group compared with the WT/LPS groups, but both groups had raised levels compared with the saline groups (Figure E3). Inflammatory Cytokines
IL-1b, TNF-a, and IL-6 play key roles in the pathogenesis of lung injury (6). The concentrations of IL-6 and TNF-a were significantly elevated in BALF from mice in the WT/LPS and TAFI KO/LPS groups compared with the saline-treated groups. The concentration of IL-6 in BALF was significantly elevated in the TAFI KO/LPS group compared the TAFI KO/SAL group. All of these inflammatory cytokines were significantly increased in the TAFI KO/LPS group compared with the WT/LPS group in BALF (Figure 3). The plasma IL-6 levels were not elevated in the WT/LPS mice, but they were significantly increased in the TAFI KO/LPS groups compared with the WT/LPS, WT/SAL, and TAFI KO/SAL groups (Figure 3), whereas there was no detectable change in the levels of IL-1b and TNF-a in plasma (data not shown). Complement System
Components of the complement system have been reported to play an important role in the pathogenesis of ARDS (19). To
confirm this in this model, we measured the concentration of the anaphylatoxin C5a in the different groups of mice in BALF and plasma. The results showed increased concentrations of C5a in BALF and plasma from WT/LPS and TAFI KO/LPS mice compared with saline-treated mice with much more significantly increased levels in the TAFI KO/LPS group than in the WT/LPS group (Figure 4).
Pathogenic Role of the Complement System
Because TAFI KO mice have exacerbated disease that suggests that TAFI or TAFIa is protective, but there are several possible substrates. To clarify if the protective role of TAFI in the mechanism of ALI is via inactivation of C5a rather than an alternative TAFIa substrate, we pretreated the mice with an antagonist of the C5a receptor two times by inhalation before inducing lung injury by LPS instillation. Before performing this experiment, the C5a receptor antagonist was confirmed to bind to C5a receptor on lung cells by in vitro (Figures E4A and EB) and in vivo (Figure E4C– E4E) experiments. The group of TAFI KO mice pretreated with the C5a receptor antagonist (TAFI KO/C5aRA/LPS) before LPS intratracheal instillation showed significantly decreased infiltration of inflammatory cells in the lungs (Figure 5); decreased lung mRNA expression of the inflammatory cytokines IL-6, monocyte chemotactic protein (MCP)-1, and IL-1b (Figures 6A and 6B); and decreased lung tissue concentration of C5a peptides in the TAFI KO/C5aRA/LPS mice compared with the untreated TAFI
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Figure 2. Protein leakage and coagulation system. Samples were harvested 24 hours after LPS instillation. (A) The concentration of total protein was significantly increased in the TAFI KO/LPS group compared with the other groups. The concentrations of thrombin–antithrombin (TAT) complex in BALF (B) and lung tissue (C) were not significantly different between the WT/LPS and TAFI KO/LPS groups, but in BALF and lung tissue there was a significant increase in both groups of LPS-treated mice (WT/LPS and TAFI KO/LPS) compared with their respective saline controls (WT/SAL and TAFI KO/SAL). (D) BALF D-dimer was significantly different between the WT/LPS and TAFI KO/LPS groups; it was decreased in WT/LPS compared with control mice but not at significant level (n ¼ 5 mice in each group). The figure shows representative results from one of three independent experiments. Bars indicate means 6 SEM. Statistical analysis by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. *P , 0.05 versus the respective saline groups; xP , 0.05 versus WT/LPS.
KO/LPS group (Figure 6C). However, there was no significant difference in the number of lung infiltrating cells (Figure 5), in lung RNA expression of MCP-1 and IL-1b, or in the lung tissue level of C5a (Figures 6A–6c) between WT mice pretreated with C5a receptor antagonist (WT/LPS/C5aRA) and those pretreated with saline (WT/LPS/SAL) before LPS instillation. To clarify this lack of response in the WT mice, we performed another experiment in which WT mice were pretreated four times by inhalation
with the C5a receptor antagonist. WT mice receiving more frequent inhalations of the C5a receptor antagonist had decreased lung cell infiltration and reduced concentrations of total protein in BALF and reduced levels of C5a, TAT, and PAI-1 in lung tissue homogenates (Figures E5A–E5E). The BALF concentrations and the lung mRNA expression level of TNF-a, MCP-1, and IL-1b were also significantly reduced in the WT/LPS/C5aRA group compared with the WT/LPS/SAL group (Figures 7A and 7B).
Figure 3. Inflammatory cytokines. Samples were harvested 24 hours after LPS instillation. The BALF and plasma concentration of IL-6 and the BALF concentration of TNF-a and IL-1b were significantly elevated in the TAFI KO/LPS groups compared with all other groups (n ¼ 5 mice in each group). The figure shows representative results from one of three independent experiments. Bars indicate means 6 SEM. Statistical analysis by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. *P , 0.05 versus the respective saline groups; xP , 0.05 versus WT/LPS.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 49 2013 Figure 4. The complement system. Samples were harvested 24 hours after LPS instillation. The BALF and plasma levels of C5a were significantly increased in the WT/LPS and TAFI KO/LPS mice compared with all saline-treated groups. The concentrations of C5a in BALF and plasma were markedly elevated in the TAFI KO/LPS groups compared with the WT/LPS group (n ¼ 5 mice in each group). The figure shows representative results from one of three independent experiments. Bars indicate means 6 SEM. Statistical analysis by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. *P , 0.05 versus the respective saline groups; xP , 0.05 versus WT/LPS.
DISCUSSION The results of this study show that the TAFI KO animals have worse disease than WT and therefore TAFI plays a protective role in the mechanism of LPS-induced ALI. Because the difference between the TAFI KO mice and WT is abrogated by a pharmacological intervention preventing C5a signaling through its cognate receptor, it is likely that the protective effect of TAFI is due to inactivation of the complement anaphalotoxins C3a and C5a. The Complement System and Lung Injury
The complement system plays a critical role in innate host defense (5, 6). Components of the complement system can directly lyse bacteria, enhance bacterial phagocytosis by opsonization, stimulate the recruitment of immune cells, and increase vascular leakage of inflammatory mediators from the bloodstream (5, 6). Excessive activation of the complement system may contribute to the pathogenesis of ALI/ARDS (19–23). Previous studies have shown that increased levels of complement proteins in the lung of patients with ARDS were associated with worse clinical outcomes (20, 22, 24). Enhanced concentration of complement factors has been
also reported in ALI induced in mice by intratracheal instillation of LPS, and these observations are consistent with the increased lung levels of C5a in our LPS-treated mice (25, 26). In addition, we demonstrated that the lung level of C5a is dose dependent, being insignificantly increased when the intratracheal dose of LPS is 100 mg but significantly high at doses of 150 or 200 mg of intratracheal LPS compared with control mice. The use of relatively low doses of LPS (50 or 100 mg) for inducing ALI may explain why previous studies found no increase in the levels of complement factors in the lungs of LPS-treated mice (16, 17). We have also shown in this study that pretreatment with inhaled C5a receptor antagonist ameliorates LPS-induced ALI in WT mice, further supporting the role of the complement system in the pathogenesis of the disease. In addition to the complement system, other mechanisms are involved in LPS-induced ALI. For example, LPS can enhance the inflammatory response in ALI by increasing the surface expression of tissue factor, the initiator of coagulation, leading to enhanced generation of thrombin, which binds to its receptor (protease-activated receptor [PAR]-1, -3, and 4) and enhances the secretion of proinflammatory cytokines and the recruitment of leukocytes (6). LPS can also bind to Toll-like receptor 4,
Figure 5. C5a receptor antagonist inhibits lung cell infiltration in TAFI KO mice with acute lung injury. WT and TAFI KO mice were pretreated with complement C5a receptor antagonist (C5aRA) (WT/C5aRA/LPS [n ¼ 4]; TAFI KO/C5aRA/LPS [n ¼ 5]) or saline (WT/SAL/LPS [n ¼ 4]; TAFI KO/SAL/LPS [n ¼ 6]) by inhalation using a nebulizer 24 hours and 2 hours before intratracheal instillation of LPS. WT/SAL (n ¼ 3) and TAFI KO/SAL (n ¼ 5) groups received saline instillation instead of LPS. Samples were harvested 24 hours after LPS instillation. Lung tissues were stained by H&E, and the number of cell nuclei per area of lung field was determined as described in MATERIALS AND METHODS. (A and B) Cell infiltration was significantly decreased in mice (TAFI KO/ C5aRA/LPS) treated with the C5a receptor antagonist compared with untreated TAFI KO mice with acute lung injury (TAFI KO/SAL/LPS). The figure shows representative results from one of two independent experiments. Bars indicate means 6 SEM. Scale bars indicate 50 mm. Statistical analysis by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. *P , 0.05 versus TAFI KO/SAL/LPS.
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Figure 6. C5a receptor antagonist treatment decreased cytokine mRNA expression in TAFI KO mice with acute lung injury. WT and TAFI KO mice were pretreated with C5aRA (WT/C5aRA/LPS [n ¼ 4]; TAFI KO/C5aRA/LPS [n ¼ 5]) or saline (WT/SAL/LPS [n ¼ 4]; TAFI KO/SAL/LPS [n ¼ 6]) by inhalation using a nebulizer 24 and 2 hours before intratracheal instillation of LPS. WT/SAL (n ¼ 3) and TAFI KO/SAL (n ¼ 5) groups received saline instillation instead of LPS. Samples were harvested 24 hours after LPS instillation. (A) After separation of PCR products by gel bands were quantitated by densitometry. (B) The lung mRNA expressions of IL-1b, IL-6, and monocyte chemotactic protein (MCP)-1 were significantly decreased in mice (TAFI KO/C5aRA/LPS) treated with C5a receptor antagonist compared with untreated TAFI KO mice with acute lung injury (TAFI KO/SAL/LPS). (C ) The lung level of C5a was significantly decreased in mice (TAFI KO/C5aRA/LPS) treated with C5a receptor antagonist compared with untreated mice (TAFI KO/SAL/LPS). The figure shows representative results from one of two independent experiments. Bars indicate the means 6 SEM. Statistical analysis by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. *P , 0.05 versus TAFI KO/LPS. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
activating intracellular pathways that increase the release of proinflammatory factors and chemotaxis of inflammatory cells into the lungs (6). In the present study, the inflammatory response and the level of C5a in the lung tissue were significantly reduced in TAFI-deficient mice with lung injury pretreated with C5aRA, supporting the critical role of TAFI-mediated regulation of CR5a in ALI. However, the fact that lung injury was not completely inhibited by the C5aRA suggests that, in addition to C5a, other mechanistic pathways are involved in the pathogenesis of LPS-induced ALI.
(6). High concentrations of proinflammatory cytokines in the lungs have been associated with increased mortality in patients with ARDS (5, 27, 28). Consistent with these observations, in the present study we observed significantly higher concentrations of IL-1b, TNF-b, and IL-6, along with increased lung infiltration of neutrophils in WT mice treated with LPS compared with control mice. These inflammatory changes were significantly much worse in LPS-treated, TAFI-deficient mice than in their WT counterparts, suggesting a protective role of TAFI in ALI.
The Inflammatory Response and TAFI
The Coagulation/Fibrinolysis Cascade and TAFI
The inflammatory response in ALI may be triggered by an ongoing lung tissue insult or by a systemic infectious process leading to increased lung and plasma concentration of cytokines, enhanced endothelial/epithelial barrier permeability with leakage of fluids, and plasma proteins into interstitial and alveolar spaces and subsequent recruitment of leukocytes, particularly of neutrophils, into the lungs (5, 6). IL-1b and TNF-a are proinflammatory cytokines secreted by alveolar macrophages during the early stages of the disease and thus are believed to be responsible for the initial pathophysiological changes in the lungs (6). Both of these proinflammatory cytokines can stimulate the secretion of chemokines, including MCP-1, macrophage inflammatory protein-1a, and IL-8, and thus they can further promote the accumulation of monocytes, lymphocytes, and neutrophils at sites of lung injury
Imbalance between the coagulation and fibrinolysis systems occurs in patients with ALI (5). There is an increased activation of the coagulation cascade with accelerated thrombin generation and impairment of fibrinolysis (5). This imbalance results in excessive cleavage of fibrinogen by thrombin and increased deposition of fibrin, which may further promote inflammation in the injured lungs (5). In our present study, we found a low level of D-dimer in WT mice treated with LPS compared with control mice, confirming impaired fibrinolytic activity in this lung injury. The high level of PAI-1 may explain the impairment of fibrinolysis in the WT mice with LPS-induced lung injury (5, 29). Interestingly, despite the higher level of PAI-1 in the TAFI KO/LPS group than in the WT/LPS group, the BALF level of D-dimer remained the same as in control mice. This finding may
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Figure 7. A high dose of C5a receptor antagonist blocked lung injury in WT mice. WT mice were pretreated with saline (WT/LPS/SAL [n ¼ 4]) or C5aRA (WT/C5aRA/LPS [n ¼ 4]) 24, 6, 4, and 2 hours before receiving intratracheal LPS. WT/Sal mice were treated with saline alone. Samples were harvested 24 hours after LPS instillation. The BALF levels (A) and the lung RNA expression (B) of TNF-a, MCP-1, and IL-1b in BALF were significantly decreased in mice treated with C5a receptor antagonist compared with untreated mice. Bars indicate means 6 SEM. Statistical analysis by ANOVA with post hoc analysis using Fisher’s predicted least significant difference test. *P , 0.05 versus WT/SAL group; xP , 0.05 versus WT/LPS/SAL.
be explained by the lack of TAFI, which is also an inhibitor of plasmin generation (8). It could be that the TAFI-deficient mice have a lower lung fibrin burden, muting the detection of increased fibrinolysis anticipated in these animals. The Complement System and TAFI
Experimental in vivo models of allergic bronchial asthma and autoimmune arthritis have demonstrated that TAFIa exerts its antiinflammatory effect by inactivating C5a (11, 12), whereas in some other models its effects are via inhibition of fibrinolysis (8). Previously it had been shown that instillation of C5a directly into the lungs of TAFI KO mice caused worse alveolitis than in WT mice (30). In this study, we hypothesized that the stronger inflammatory response in LPS-treated, TAFI-deficient mice than in their WT counterparts is due to increased C5a activity generated endogenously. To demonstrate this thesis, we pretreated mice with C5a receptor antagonist before LPS intratracheal instillation and compared the results with untreated mice. The results showed a reduction in the expression of inflammatory cytokines and in the level of C5a in the lungs of LPS-treated, TAFI-deficient mice treated with inhaled C5aRA compared with untreated mice. Unfortunately, the
available ELISAs for C5a measure total C5a without distinguishing between active C5a and inactive C5adesArg produced by TAFIa cleavage of C5a. We speculate that the reduction in C5a level by inhaled C5aRA therapy in TAFI-deficient mice is due to mitigation of the vicious cycle of C5a-mediated cell receptor activation, inflammatory response, and complement activation with C5a generation. Taken together these data suggest that the antiinflammatory activity of TAFI is mediated by inactivation of C5a. Conclusions
The results of the present investigation showed that TAFI is protective in ALI, suggesting that TAFI plays a critical role in the pathogenesis of the disease via its regulation of the activity of complement C5a. Author disclosures are available with the text of this article at www.atsjournals.org.
References 1. Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, Camporota L, Slutsky AS. Acute respiratory distress syndrome: the Berlin definition. JAMA 2012;307:2526–2533.
Naito, Taguchi, Kobayashi, et al.: TAFI in ALI 2. Villar J, Blanco J, Anon JM, Santos-Bouza A, Blanch L, Ambros A, Gandia F, Carriedo D, Mosteiro F, Basaldua S, et al. The alien study: incidence and outcome of acute respiratory distress syndrome in the era of lung protective ventilation. Intensive Care Med 2011;37:1932–1941. 3. Walkey AJ, Summer R, Ho V, Alkana P. Acute respiratory distress syndrome: epidemiology and management approaches. Clin Epidemiol 2012;4:159–169. 4. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, Stern EJ, Hudson LD. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–1693. 5. Cross LJ, Matthay MA. Biomarkers in acute lung injury: insights into the pathogenesis of acute lung injury. Crit Care Clin 2011;27:355–377. 6. Matthay MA, Zemans RL. The acute respiratory distress syndrome: pathogenesis and treatment. Annu Rev Pathol 2011;6:147–163. 7. Colucci M, Semeraro N. Thrombin activatable fibrinolysis inhibitor: at the nexus of fibrinolysis and inflammation. Thromb Res 2012;129:314–319. 8. Morser J, Gabazza EC, Myles T, Leung LL. What has been learnt from the thrombin-activatable fibrinolysis inhibitor-deficient mouse? J Thromb Haemost 2010;8:868–876. 9. Heylen E, Willemse J, Hendriks D. An update on the role of carboxypeptidase U (TAFIa) in fibrinolysis. Front Biosci 2011;16:2427–2450. 10. Campbell WD, Lazoura E, Okada N, Okada H. Inactivation of c3a and c5a octapeptides by carboxypeptidase r and carboxypeptidase n. Microbiol Immunol 2002;46:131–134. 11. Fujiwara A, Taguchi O, Takagi T, D’Alessandro-Gabazza CN, Boveda-Ruiz D, Toda M, Yasukawa A, Matsushima Y, Miyake Y, Kobayashi H, et al. Role of thrombin-activatable fibrinolysis inhibitor in allergic bronchial asthma. Lung 2012;190:189–198. 12. Song JJ, Hwang I, Cho KH, Garcia MA, Kim AJ, Wang TH, Lindstrom TM, Lee AT, Nishimura T, Zhao L, et al. Plasma carboxypeptidase b downregulates inflammatory responses in autoimmune arthritis. J Clin Invest 2011;121:3517–3527. 13. Finch AM, Wong AK, Paczkowski NJ, Wadi SK, Craik DJ, Fairlie DP, Taylor SM. Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor c5a. J Med Chem 1999;42:1965–1974. 14. Paczkowski NJ, Finch AM, Whitmore JB, Short AJ, Wong AK, Monk PN, Cain SA, Fairlie DP, Taylor SM. Pharmacological characterization of antagonists of the c5a receptor. Br J Pharmacol 1999;128:1461–1466. 15. Nagashima M, Yin ZF, Zhao L, White K, Zhu Y, Lasky N, Halks-Miller M, Broze GJ Jr, Fay WP, Morser J. Thrombin-activatable fibrinolysis inhibitor (tafi) deficiency is compatible with murine life. J Clin Invest 2002;109:101–110. 16. Burrell R, Lantz RC, Hinton DE. Mediators of pulmonary injury induced by inhalation of bacterial endotoxin. Am Rev Respir Dis 1988;137:100–105. 17. Rittirsch D, Flierl MA, Day DE, Nadeau BA, McGuire SR, Hoesel LM, Ipaktchi K, Zetoune FS, Sarma JV, Leng L, et al. Acute lung injury induced by lipopolysaccharide is independent of complement activation. J Immunol 2008;180:7664–7672.
653 18. Kruithof EK. Regulation of plasminogen activator inhibitor type 1 gene expression by inflammatory mediators and statins. Thromb Haemost 2008;100:969–975. 19. Kambas K, Markiewski MM, Pneumatikos IA, Rafail SS, Theodorou V, Konstantonis D, Kourtzelis I, Doumas MN, Magotti P, Deangelis RA, et al. C5a and tnf-alpha up-regulate the expression of tissue factor in intra-alveolar neutrophils of patients with the acute respiratory distress syndrome. J Immunol 2008;180:7368–7375. 20. Hammerschmidt DE, Weaver LJ, Hudson LD, Craddock PR, Jacob HS. Association of complement activation and elevated plasma-c5a with adult respiratory distress syndrome: pathophysiological relevance and possible prognostic value. Lancet 1980;1:947–949. 21. Pittet JF, Mackersie RC, Martin TR, Matthay MA. Biological markers of acute lung injury: prognostic and pathogenetic significance. Am J Respir Crit Care Med 1997;155:1187–1205. 22. Solomkin JS, Cotta LA, Satoh PS, Hurst JM, Nelson RD. Complement activation and clearance in acute illness and injury: evidence for c5a as a cell-directed mediator of the adult respiratory distress syndrome in man. Surgery 1985;97:668–678. 23. Zimmerman JL, Dellinger RP, Straube RC, Levin JL. Phase i trial of the recombinant soluble complement receptor 1 in acute lung injury and acute respiratory distress syndrome. Crit Care Med 2000;28: 3149–3154. 24. Allegretti M, Moriconi A, Beccari AR, Di Bitondo R, Bizzarri C, Bertini R, Colotta F. Targeting c5a: recent advances in drug discovery. Curr Med Chem 2005;12:217–236. 25. Bolger MS, Ross DS, Jiang H, Frank MM, Ghio AJ, Schwartz DA, Wright JR. Complement levels and activity in the normal and lps-injured lung. Am J Physiol Lung Cell Mol Physiol 2007;292:L748–L759. 26. Rabinovici R, Yeh CG, Hillegass LM, Griswold DE, DiMartino MJ, Vernick J, Fong KL, Feuerstein G. Role of complement in endotoxin/ platelet-activating factor-induced lung injury. J Immunol 1992;149: 1744–1750. 27. Meduri GU, Headley S, Kohler G, Stentz F, Tolley E, Umberger R, Leeper K. Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS: plasma IL-1 beta and IL-6 levels are consistent and efficient predictors of outcome over time. Chest 1995;107:1062–1073. 28. Pugin J, Ricou B, Steinberg KP, Suter PM, Martin TR. Proinflammatory activity in bronchoalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1. Am J Respir Crit Care Med 1996; 153:1850–1856. 29. Sebag SC, Bastarache JA, Ware LB. Therapeutic modulation of coagulation and fibrinolysis in acute lung injury and the acute respiratory distress syndrome. Curr Pharm Biotechnol 2011;12: 1481–1496. 30. Nishimura T, Myles T, Piliponsky AM, Kao PN, Berry GJ, Leung LL. Thrombin-activatable procarboxypeptidase b regulates activated complement c5a in vivo. Blood 2007;109:1992–1997.
