INTRODUCTION

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The interstitial lung diseases (ILD) comprise a diverse group of disorders characterized by inflammatory changes of the interstitial and alveolar compartments of the lung with mesenchymal cell proliferation, extracellular matrix deposition, and scarring that result ultimately in progressive loss of normal lung tissue (1). The common physiopathological process of all ILD is the occurrence of epithelial/endothelial injury. Oxidants, immunological and inflammatory factors, or viral infections have been proposed as the initial insults in ILD (1). Alveolar injury results in transudation of plasma and activation of the intraalveolar coagulation mechanisms leading to the formation of a fibrin-rich intraalveolar exudate (2). It is believed that this normal procoagulant activity of bronchoalveolar surface limits the extent of alveolar hemorrhage following lung injury (3). However, several studies suggested that excessive procoagulant activity and abnormal fibrin turnover in the alveolar space may play relevant roles in the pathogenesis of acute lung injury and ILD. Interstitial and intraalveolar fibrin, its degradation products, and local thrombin generation may potentiate the acute inflammatory response by their effects on chemotaxis, vascular permeability, and immunomodulation (4, 5). Thrombin, fibrin, and its degradation products have been shown to impair surfactant function, decrease lung compliance, and reduce gas exchange (6, 7). In addition, these components of the coagulation process may have important influence on healing and on the course of pulmonary fibrosis. For example, fibroblasts may aggregate upon fibrin matrices which may stimulate fibroblast proliferation and promote increased local collagen deposition (8). Similarly, thrombin may promote cell adhesion and induce fibroblast and smooth muscle cell proliferation (9). Sequential pathologic evaluations have shown that the sites of early fibrin deposition correlate with the location of the subsequent fibrotic process in the acute respiratory distress syndrome and idiopathic pulmonary fibrosis (IPF), further supporting the role of the coagulation pathways in the process of pulmonary fibrosis (10, 11).

Hypercoagulability results from the disruption of the balance between the procoagulant and anticoagulant factors that control clotting homeostasis. This imbalance may occur as a result of an ongoing stimulus to coagulation or a defect of the natural anticoagulant or fibrinolytic process. The basic mechanisms that regulate clotting activation within the intravascular compartment have been reasonably well defined. The most important anticoagulant system within the intravascular space constitutes the protein C (PC) pathway (12). PC is a vitamin K-dependent plasma glycoprotein that, after being proteolytically cleaved by thrombin complexed to thrombomodulin on endothelial or platelet surface, is converted to activated protein C (APC) which is the enzyme effector of the PC anticoagulant system (13). APC exerts anticoagulant activity by inactivating factor Va and factor VIIIa on the membrane of platelets and endothelial cells, and also, by stimulating fibrinolysis in concert with protein S (13). The main inhibitor of APC is PC inhibitor (PCI) with which it forms the APC-PCI complex (14). The clinical importance of the protein C pathway for the regulation of the coagulation system is illustrated by the frequency of thromboembolic disease in subjects with heterologous or homologous deficiency of PC or protein S and the recently described resistance to APC (15). No study has been previously carried out to evaluate the levels of PC and APC- PCI in the bronchoalveolar lavage fluid (BALF) and plasma of patients with ILD.

In the present study, to gain some insights into the role of the PC anticoagulant system in ILD, we assessed the levels of molecular markers of the PC anticoagulant system in BALF and plasma of patients with sarcoidosis, IPF, and ILD associated with collagen vascular disorders (CVD-ILD).

	METHODS

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Patient Population

This study comprised 41 newly diagnosed patients with ILD. There were 11 patients (10 male, 1 female) with IPF, 14 (7 male, 7 female) with sarcoidosis, and 16 (3 male, 13 female) with CVD-ILD. Among this latter group, there were two patients with, each, rheumatoid arthritis, systemic sclerosis, or systemic lupus erythematosus, three with dermatomyositis, and seven with mixed connective tissue disease. The diagnosis of IPF was based on the presence of unexplained dyspnea, progressive bilateral pulmonary infiltrates, and evidence of pulmonary fibrosis in histologic specimens obtained by transbronchial or open lung biopsy (18). The diagnosis of sarcoidosis was established by histologic evidence of noncaseating granulomas in transbronchial lung biopsy specimens and compatible clinical, serological, and radiographic findings (19). The diagnosis of CVD-ILD was based on previously reported criteria for each group of CVD and the histologic evidence of pulmonary fibrosis in transbronchial lung biospy specimens (20). Six healthy nonsmoking volunteers served as control subjects. Written informed consent was obtained from all subjects. The study protocol was approved by the Mie University Hospital Institutional Review Board and it was carried out following the principles of the Helsinski Declaration.

Sampling of Blood and Bronchoalveolar Lavage

Bronchoalveolar lavage was performed as previously described (21). A fiberoptic bronchoscope (Olympus 1T10; Olympus, Tokyo, Japan) was wedged into a segmental bronchus of the middle lobe or lingula. Four 50-ml aliquots of warmed sterile normal saline were instilled and retrieved immediately by suction at low negative pressure adjusted by a vacuum regulator. The pooled BALF was collected on ice and processed within 1 h. The total amount of recovered fluid was measured and the recovery rate was calculated. The fluid was filtered through two layers of sterile gauze and centrifuged at 150 g for 10 min at 4° C. The cell-free supernatant was separated and stored in small aliquots at 80 ° C until analysis. The cellular fraction was resuspended in RPMI 1640 medium (Celox Laboratories, Roseville, MN) and total cells were counted using a Neubauer hemocytometer counting chamber. Differential cell count was carried out in a Giemsa-stained cytocentrifuge preparation at ×1,000 magnification. Blood samples were drawn from an anticubital vein and poured in vacutainers containing 3.8% sodium citrate. After centrifuging at 500 g for 20 min at 4° C, the plasma samples were stored in aliquots at 80° C until use.

Laboratory Measurements

The level of thrombin-antithrombin III complex (TAT), a sensitive marker of coagulation activation, was measured using a commercially available immunoassay kit (Enzygnost-TAT; Behrinwerke A.G., Marburg, Germany) following the manufacturer's instructions. The intra-assay and interassay precisions of the TAT assay were both less than 10%. BALF and plasma concentrations of PC were determined by a solid-phase immunoassay using a human polyclonal anti-PC and biotin-labeled monoclonal anti-PC antibodies following previously described methods; the anti-PC monoclonal antibody binds specifically to PC but not to the APC component of the APC-PCI complex (22). PC values were extrapolated from a curve drawn using standard concentrations of PC. The intra-assay and the interassay coefficients of variation for PC were 5% and 6%, respectively. The BALF and plasma levels of APC-PCI complex, a marker of ongoing PC activation, were measured by an enzyme-linked immunoassay as described (22). Briefly, monoclonal anti-PCI (3 µg/ml) was coated on microtiter plate by overnight incubation. BALF or blood samples were prepared as follows: aliquots of BALF or plasma were treated with 3.8% sodium citrate and then with 10 mM barium chloride. This mixture was then centrifuged at 11,000 g for 5 min. The precipitate dissolved in washing buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.5 mM EDTA, 0.1% Tween 20, and 0.5% bovine serum albumin) was then incubated overnight in anti-PCI-antibody-coated microtiter plate after appropriate washing and blocking of nonspecific bindings. After washing, peroxidase-conjugated monoclonal anti-PC antibody (0.5 µg/ml) was added to each microtiter well and incubated for 5 h. After appropriate washing, the peroxidase substrate, 3,3',5,5' tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD), was added to each well and absorbance was measured at 450 nm. APC- PCI values were extrapolated from a curve drawn using standard concentrations of the complex. The interassay and the intra-assay coefficients of variability were 5% and 7%, respectively. BALF concentrations of PCI were determined by a solid-phase immunoassay using human monoclonal anti-PCI and peroxidase-conjugated monoclonal anti-PCI antibodies. The interassay and the intra-assay coefficients of variability were both less than 10%. Protein concentration in BALF was measured by the Bradford's method using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) (23).

Statistical Analysis

Data are expressed as the mean ± SE unless otherwise specified. The statistical difference between two variables was calculated using Wilcoxon's rank test, and Kruskal-Wallis analysis of variance was used for calculating the difference among the means of three or more variables. The strength of correlation between variables was calculated by the Pearson-product moment correlation or by the Spearman's correlation according to the distribution of the data. Statistical analyses were carried out using the StatView 4.1 package software (ABACUS CONCEPTS, Berkeley, CA) for Macintosh. Values of p < 0.05 were considered as statistically significant.

	RESULTS

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Cellular Analysis, Total Protein, and TAT in BALF

As depicted in Table 1, the total protein concentration in BALF was significantly higher in patients with sarcoidosis and CVD-ILD than in control subjects. The total cell count in patients with CVD-ILD, the percentage of alveolar macrophages and lymphocytes in those with sarcoidosis, and the percentage of neutrophils in those with IPF were significantly increased in BALF as compared with control subjects.

The levels of TAT in BALF were significantly higher (p < 0.05) in patients with sarcoidosis (1.78 ± 0.11 µg/L) and CVD-ILD (1.69 ± 0.18 µg/L) than in normal healthy control subjects (1.34 ± 0.04 µg/L). Concentrations of TAT in BALF also tended to be higher in patients with IPF (1.60 ± 0.11 µg/L) than in control subjects. BALF concentration of TAT was significantly correlated with concentrations of total protein (r = 0.8; p < 0.001) and PC (r = 0.6; p < 0.02) in all patients (n = 41) with ILD.

Components of PC Pathway in BALF

Antigen levels of PC in BALF (Figure 1) were significantly higher in patients with IPF (610 ± 150 ng/ml), sarcoidosis (680 ± 170 ng/ml), and CVD-ILD (1,580 ± 600 ng/ml) than in healthy control subjects (230 ± 140 ng/ml). Concentrations of APC-PCI complex in BALF (Figure 2) were significantly decreased in patients with IPF (0.46 ± 0.16 ng/ml), sarcoidosis (0.43 ± 0.11 ng/ml), and CVD-ILD (0.50 ± 0.15 ng/ml) as compared with healthy control subjects (1.08 ± 0.23 ng/ml). As another index of the degree of PC activation in the alveolar space, the APC-PCI/PC ratio was calculated. The APC- PCI/PC ratios (Figure 3) were markedly lower in patients with IPF (2.70 ± 1.74 ng/µg), sarcoidosis (1.94 ± 0.0.82 ng/µg), and CVD-ILD (1.89 ± 0.68 ng/µg) than in healthy controls (15.91 ± 8.45 ng/µg). Concentrations of PCI in BALF tended to be higher in patients with IPF (0.06 ± 0.03 µg/ml), sarcoidosis (0.29 ± 0.19 µg/ml), and CVD-ILD (0.08 ± 0.03 µg/ml) as compared with healthy control subjects (0.04 ± 0.01 µg/ml).

View larger version (12K): [in this window] [in a new window]   Figure 1.   PC antigen levels in BALF from ILD patients and control subjects. Antigen levels of PC were significantly higher in patients with IPF, sarcoidosis, and CVD-ILD than in healthy control subjects. *p < 0.05 versus control. Data are mean ± SE.

View larger version (13K): [in this window] [in a new window]   Figure 2.   APC-PCI levels in BALF from ILD patients and control subjects. The concentrations of APC-PCI complex were significantly decreased in patients with IPF, sarcoidosis, and CVD-ILD as compared with normal healthy control subjects. *p < 0.05 versus control. Data are mean ± SE.

View larger version (12K): [in this window] [in a new window]   Figure 3.   APC-PCI/PC ratio in BALF from ILD patients and control subjects. The APC-PCI/PC ratios were markedly lower in patients with IPF, sarcoidosis, and CVD-ILD than in healthy control subjects. *p < 0.05 versus control. Data are mean ± SE.

Plasma Levels of Clotting Markers

Plasma levels of TAT, PC, APC-PCI complex, and the values of APC-PCI/PC ratio are described in Table 2. Plasma concentrations of TAT were significantly increased in patients with IPF and CVD-ILD, and those of PC in patients with CVD-ILD as compared with the control group. The plasma concentration of APC-PCI and the APC-PCI/PC ratio tended to be lower in all ILD patients, particularly in those with CVD-ILD, than in healthy control subjects.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study showed that there is an increased procoagulant activity in the alveolar space of patients with sarcoidosis, IPF, and CVD-ILD. Previous studies suggested that enzymes mediating clotting and fibrinolysis may play important functions in the normal physiology of both the vascular and alveolar compartments of the lung (24). During the process of acute lung injury, intraalveolar transudation of plasma proteins occurs and several proinflammatory stimuli such as cytokines or endotoxin induce the expression of tissue factor by the capillary endothelium, alveolar epithelial cells, or macrophages (25). Tissue factor activates the extrinsic pathway of the coagulation system on the surface of these cells leading to intraalveolar deposition of fibrin (27). Alternatively, activation of the intrinsic pathway of coagulation, involving factors XII, XI, IX, VIII, prekallikrein, and high-molecular-weight kallikrein, may also occur in the alveolar compartment of the lung. It is believed that fibrin formation in the alveolar space may serve to control excessive local hemorrhage and to provide a temporary structural support to injured lung tissues (3). Thrombin generation and local fibrin deposits may also contribute to immobilization and focal accumulation of alveolar macrophages that are important for local clearance and host defense mechanisms (28). Locally generated thrombin may also stimulate the proliferation of other lung cells (28). In the present study, the increased intraalveolar concentrations of TAT observed in patients with IPF, sarcoidosis, and CVD-ILD as compared with healthy subjects also support the idea that enhanced activation of the coagulation system occurs in the intraalveolar space of patients with ILD.

The anticoagulant pathway involving PC, PS, and thrombomodulin constitutes an important modulator of coagulation mechanisms in the intravascular space (12, 13). PC is activated to APC by thrombin or alternatively by activated factor X in the presence of the membrane-bound glycoprotein, thrombomodulin (13). In the intraalveolar compartment, platelets, which express thrombomodulin, may provide the phospholipid surface for effective APC generation (29). However, the presence of platelets or the formation of thrombomodulin/ thrombin complexes in the intraalveolar space has not been as yet demonstrated. APC exerts anticoagulant activity by degrading proteolytically the active forms of both factor VIII and factor V. The presence of PC in BALF has not been previously evaluated. In this study, we demonstrated for the first time that PC antigens are also present in BALF from normal subjects. PC (Mr 62,000), which normally circulates in human plasma at a concentration of 4 µg/ml, is synthesized in the liver and endothelial cells (12). The source of PC in the alveolar compartment is unknown, but it might probably leak from the pulmonary circulation. In the present investigation, PC was also detected in BALF from patients with ILD; its concentration was markedly increased as compared with normal control subjects, and it was significantly correlated with TAT, a marker of ongoing clotting activation. These observations suggest that the PC anticoagulant system may also play a relevant biological role in the regulation of intraalveolar coagulative processes occurring in ILD.

Once the clotting system is activated in the alveolar compartment, thrombin converts fibrinogen into fibrin clots (27). The deposition of fibrin in the intraalveolar space is, under normal conditions, rapidly cleared by fibrinolytic mechanisms. This local fibrin clearance occurs because the bronchoalveolar fluid from normal individuals contains the plasminogen activator, urokinase, which converts plasminogen into plasmin, the active enzyme of the fibrinolytic system (24). Macrophages and alveolar epithelial cells are the source of urokinase (24). The activity of the plasminogen-plasmin system in the alveolar compartment is regulated by the local availability of the urokinase receptor and the plasminogen activator inhibitor-1 (30, 31). APC, the effector enzyme of the anticoagulant PC system, besides its anticoagulant function, may also affect fibrinolysis activity (32). APC may stimulate fibrinolysis, at least in part, by its ability to form a complex with or to degrade plasminogen activator inhibitor, thereby reducing the amount of this inhibitor available to constrain the generation of the fibrinolytic enzyme plasmin (33). In the present study, the levels of APC-PCI complex and the APC-PCI/PC ratio in BALF in all groups of patients with ILD, and their plasma values in those with CVD-ILD, were found to be significantly decreased as compared with normal control subjects, suggesting that decreased PC activation occurs in ILD patients. Impairment in the clearance of intraalveolar fibrin has been reported in patients with ILD as well as in animal models of acute and chronic lung injury (34, 35). Persistent fibrin deposits in the alveolar compartment form a fertile environment that may promote the growth of fibroblasts and collagen deposition leading finally to the development of pulmonary fibrosis (8). Based on these findings, it is conceivable that decreased PC activation may cause increased procoagulant activity and decreased fibrinolysis in ILD, and thus promote intraalveolar fibrosis in these disorders.

In summary, this study showed, for the first time, that decreased PC activation with increased procoagulant activity occur in patients with ILD. Further studies must be carried out to clarify the source of components of PC pathway in the intraalveolar space of these patients.