Published ahead of print on November 10, 2006, doi:10.1165/rcmb.2005-0425OC
© 2007 American Thoracic Society DOI: 10.1165/rcmb.2005-0425OC Novel Role of the Human Alveolar Epithelium in Regulating Intra-Alveolar CoagulationDivision of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; Cardiovascular Research Institute; and Departments of Medicine and Anesthesia, University of California, San Francisco, California Correspondence and requests for reprints should be addressed to Lorraine B. Ware, M.D., T1218 MCN, 1161 21st Avenue S, Nashville, TN 37232-2650. E-mail: lorraine.ware{at}vanderbilt.edu
Intra-alveolar fibrin deposition is a common response to localized and diffuse lung infection and acute lung injury (ALI). We hypothesized that the alveolar epithelium modulates intra-alveolar fibrin deposition through activation of protein C. Our obejctives were to determine whether components of the protein C activation pathway are present in the alveolar compartment in ALI and whether alveolar epithelium is a potential source. In patients with ALI, pulmonary edema fluid levels of endothelial protein C receptor (EPCR) were higher than plasma, suggesting a source in the lung. To determine whether alveolar epithelial cells are a potential source, protein C activation by A549, small airway epithelial, and primary human alveolar epithelial type II cells was measured. All three cell types express thrombomodulin (TM) and EPCR, and activate protein C on the cell surface. Activation of protein C was inhibited by cytomix (TNF-  , IL-1 , and IFN- ). Release of EPCR and TM into the conditioned medium was inhibited by the metalloproteinase inhibitors tumor necrosis factor protease inhibitor (TAPI) and GM6001, indicating that the shedding of EPCR and TM from the alveolar epithelium is mediated by a metalloproteinase. These findings provide new evidence that the alveolar epithelium can modulate the protein C pathway and thus could be an important determinant of alveolar fibrin deposition. Local fibrin deposition may be a fundamental mechanism for the lung to localize and confine injury, thus limiting the risk of dissemination of injury or infection to the systemic circulation.
Key Words: alveolar epithelium • endothelium • protein C • coagulation • acute lung injury
The alveolar epithelium plays a fundamental role in the pulmonary response to acute lung injury (1). The critical functions of the alveolar epithelium include active ion transport for the removal of alveolar edema fluid (2); secretion of surfactant to maintain alveolar stability, promote gas exchange, and assist in host defense (3); and barrier properties that protect against alveolar flooding and prevent the translocation of cytokines and bacteria into the circulation. However, the role of the alveolar epithelium in regulating intra-alveolar coagulation has not been adequately appreciated. In patients with acute lung injury, intra-alveolar activation of the coagulation cascade with the deposition of fibrin along the injured alveolar surface (hyaline membranes) has been well described (4–6). However, the mechanisms that modulate intra-alveolar fibrin deposition are not well understood. Therefore, this study explores the possible contribution of the alveolar epithelium to modulation of intra-alveolar coagulation using both in vitro studies and clinical samples from patients with acute lung injury. Identifying the cellular and molecular mechanisms that regulate intra-alveolar fibrin deposition in acute lung injury and the role of the alveolar epithelium in this process may lead to a better understanding of the pathogenesis of hyaline membrane formation. The protein C pathway is a central endogenous regulator of blood coagulation. Protein C is a vitamin K–dependent plasma glycoprotein that circulates as a biologically inactive zymogen (7). The zymogen is converted into activated protein C (APC) by the thrombomodulin (TM)–thrombin complex on the cell surface (8). Activation of protein C is further enhanced when protein C is bound to the endothelial protein C receptor (EPCR) (9). APC suppresses thrombin formation by proteolytically inactivating coagulation factors Va and VIIIa. TM and EPCR were originally described in endothelial cells (10, 11), but subsequently were also detected in several other cell types (12–14), including epithelial cells. The alveolar epithelium has a large surface area, and thus is uniquely situated to actively modulate the intra-alveolar environment. We previously reported that A549 cells and primary isolates of human alveolar type II cells release soluble TM into the medium (15). Others have shown that airway epithelial cells in culture can express protein C, EPCR, and TM, and that protein C was activated by these cells in the presence of thrombin. These protein C pathway components were down-regulated by proinflammatory cytokines (16). The findings in these studies suggest that the alveolar epithelium could play a role in modulating intra-alveolar coagulation and inflammation through the protein C pathway similar to the well-described role of the endothelium in modulating intravascular coagulation.
We have previously reported that soluble TM levels are much higher in the alveolar compartment than in simultaneous plasma samples in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), suggesting a local source in the lung (15). To test whether soluble EPCR is also high in the alveolar compartment, we measured levels of EPCR in simultaneous pulmonary edema fluid and plasma samples from patients with ALI/ARDS and found higher levels in the edema fluid, suggesting an intra-alveolar source. We then tested whether alveolar epithelial cells in culture can express TM and EPCR, and therefore activate protein C. A549 cells, human small airway epithelial cells, and primary isolates of human alveolar epithelial cells were able to activate protein C and express TM and EPCR. Finally, we hypothesized that stimulation of alveolar epithelial cells with proinflammatory cytokines would lead to a shift from an anticoagulant to a procoagulant phenotype in alveolar epithelial cells as measured by a reduction in the ability of these cells to activate protein C coupled with shedding of TM and EPCR from the cell surface. Exposure to cytomix (TNF-
Patients Patients were selected from the University of California Moffitt-Long and San Francisco General Hospital intensive care unit patients who had pulmonary edema fluid and plasma obtained between 1981 and 1998. Inclusion criteria included ALI/ARDS (19) or hydrostatic pulmonary edema (15) and aspiration of pulmonary edema fluid within 1 h of endotracheal intubation. Eligibility for inclusion in the study was based solely on availability of an adequate stored volume of plasma and edema fluid for measurement of EPCR levels. To minimize selection bias, clinical data were not made available to the investigator who chose the samples for measurement of the EPCR levels, other than the etiology of acute pulmonary edema. Patient characteristics are shown in Table 1. The Committee for Human Research at University of California, San Francisco approved this study. To collect edema fluid, a tracheal suction catheter was advanced through the endotracheal tube into a wedged position, and gentle suction was applied. A simultaneous plasma sample was obtained. Plasma samples were also obtained from 10 healthy volunteers. Edema fluid and plasma were centrifuged (3,000 x g, 10 min) and stored at –70°C.
Isolation of Human Alveolar Epithelial Cells Alveolar epithelial type II cells were isolated as previously described (15, 20, 21). Cell viability was assessed by trypan blue exclusion. Purity was > 90% (15, 20, 21). Freshly isolated type II cells were suspended in cell preservation fluid and maintained in liquid nitrogen.
Cell Culture and Treatment
Protein C Activation
Enzyme Immunoassay
Total RNA Isolation and Real-Time PCR
Statistical Analysis All findings are representative of at least two separate experiments that gave qualitatively and quantitatively similar results. Data are expressed as median (interquartile range) or mean ± SD as appropriate. Normally distributed data were analyzed using Student's t test or ANOVA with post hoc Tukey's test. Nonparametric data were analyzed using Mann Whitney U test or Spearman's correlation coefficient as appropriate. A P value of  0.05 was considered significant.
Levels of Soluble EPCR in Edema Fluid and Plasma from Human Subjects with ALI/ARDS Levels of sEPCR in the undiluted pulmonary edema fluid were 2-fold higher in patients with ALI/ARDS than in a control group of patients with hydrostatic pulmonary edema (median 94.2 ng/ml [interquartile range {IQR}, 43.5–148.7] versus median 48.5 [IQR, 22.2–94.8], P = 0.028) (Figure 1A). When simultaneous edema fluid and plasma samples were compared, levels in the pulmonary edema fluid were higher than the plasma in patients with ALI/ARDS (edema fluid to plasma ratio of 1.46 ± 1.28 in ALI/ARDS versus 0.59 ± 0.37 in hydrostatic edema, P = 0.007), suggesting an intra-alveolar source in the ALI/ARDS group. Among the patients with ALI/ARDS, levels of both lung edema fluid and plasma EPCR were associated with severity of illness as measured by the Simplified Acute Physiology Score II (SAPSII) score. For patients with SAPSII  45 (n = 18), plasma and edema fluid EPCR levels were 118 (IQR, 66–175) ng/ml and 135 ng/ml (IQR, 43–261), respectively (Figures 1B and 1C). By comparison, for patients with SAPSII < 45 (n = 15), plasma and edema fluid levels were 61 ng/ml (IQR, 36–108) (P = 0.013) and 72 ng/ml (IQR, 42–108) (P = 0.058), respectively (Figures 1B and 1C). We also compared levels of soluble thrombomodulin and soluble EPCR in the pulmonary edema fluid from patients with ALI/ARDS. Some of the thrombomodulin levels have been previously published (15). There was a strong correlation between soluble thrombomodulin and EPCR levels (r = 0.73, P < 0.001) (Figure 1D), suggesting that they may be released into the alveolar compartment in the acutely injured lung by a common mechanism.
Thrombomodulin and EPCR in Alveolar Epithelial Cells: Effect of Proinflammatory Cytokines TM and EPCR proteins were detected in the cell lysates and conditioned medium from unstimulated A549 cells (Figures 2A and 2B), human small airway epithelial cells (Figure 2C), and primary isolates of human alveolar epithelial type II cells (Figure 2D). A549 cells were then exposed to 20 ng/ml cytomix (TNF- , IL-1, IFN-) for 3–24 h or to varying doses from 1–50 ng/ml for 18 h. Exposure to cytomix led to a time- and dose-dependent release of thrombomodulin and EPCR into the conditioned medium by A549 cells (Figures 2A and 2B). After 24 h exposure to 20 ng/ml cytomix, TM protein levels had risen approximately 4-fold compared with 0 h. However, when the sum of cell lysate and conditioned medium levels was considered, there was no difference in total TM protein levels per well with or without cytomix exposure (data not shown), suggesting that the change in levels in the conditioned medium did not reflect new protein synthesis. Similarly, after 24 h exposure to 20 ng/ml cytomix, EPCR protein levels had risen approximately 3-fold compared with 0 h. When cell lysates and conditioned medium levels of EPCR for each well were combined, there was no difference among total EPCR antigen levels per well with or without cytomix exposure (data not shown), again suggesting that the change in levels in the conditioned medium did not reflect new protein synthesis. A similar time- and dose-dependent release of TM and EPCR into the conditioned medium was observed in human SAECs and primary human alveolar epithelial cells (Figures 2C and 2D).
Activation of Protein C by Alveolar Epithelial Cells To determine whether the TM and EPCR that were detected in alveolar epithelial cells were functionally active, we determined whether alveolar epithelial cells could activate protein C in the presence of thrombin. A549 cells (Figures 3A and 3B), human SAECs (Figure 3C) and primary human type II cells (Figure 3D) all generated APC in the presence of thrombin and protein C. To compare to a cell type known to activate protein C, human umbilical vein endothelial cells were used in the same system. There was no significant difference between the quantity of activated protein C generated by similar numbers of A549 cells and HUVECs (data not shown). Generation of APC by A549 cells declined significantly with increasing length of exposure to cytomix (Figure 3A). The fall in protein C activation was also dose dependent (Figure 3B). There was also a significant dose-dependent decrease in protein C activation in human SAECs and primary human type II epithelial cells after exposure to cytomix (Figures 3C and 3D).
Expression of TM and EPCR by Alveolar Epithelial Cells Both the alveolar epithelial cell line (A549) (Figure 4) and primary isolates of human type II cells (not shown) expressed TM and EPCR mRNA. Real-time PCR demonstrated no significant changes in levels of TM or EPCR mRNA expression after cytomix treatment for up to 24 h in A549 cells.
Mechanism of TM and EPCR Antigen Release in Response to Proinflammatory Cytokines Previous studies in endothelial cells suggest that EPCR shedding is a metalloproteolytic process and that TM shedding is modulated by neutrophils and their release products (22, 23). To investigate the mechanism of TM and EPCR shedding from alveolar epithelial cells in response to cytomix, we tested the influence of metalloproteinase inhibitors on this process. TAPI and GM6001 are hydroxamic-based inhibitors of metalloproteinases. In A549 cells, both TAPI and GM6001 significantly inhibited the shedding of TM and EPCR that was induced by cytomix (Figure 5A). Since cell surface TM and EPCR are essential to PC activation, we hypothesized that the inhibition of TM and EPCR shedding might restore the activation of protein C on the epithelial cell surface. To test this hypothesis, we measured activation of protein C in the presence of TAPI and GM6001 after cytomix exposure. Activation of protein C was partially restored by TAPI and GM 6001 (Figure 5B). To further define the possible metalloproteinase that mediates EPCR shedding, we measured EPCR shedding after cytomix stimulation for 18 h in the presence or absence of the metalloproteinase inhibitors TIMP-1, -2, or -3 (5 µg/ml). The modest and nonspecific inhibitory pattern for shedding of both TM and EPCR observed with the TIMPs (Figure 5C) along with the prolonged time course of cytomix-mediated release was not consistent with rapid up-regulation of a sheddase such as ADAM-17, but rather suggests that a matrix metalloproteinase may be involved.
The cellular and molecular mechanisms that govern intra-alveolar fibrin deposition in ALI/ARDS are unknown. The major findings of our study can be summarized as follows. First, undiluted pulmonary edema fluid from patients with ALI/ARDS contains soluble EPCR in levels higher than plasma, suggesting a local source in the lung. Second, alveolar epithelial cells express active TM and EPCR, and can activate protein C on the cell surface. Third, TM and EPCR shedding is induced and protein C activation is inhibited by exposure to cytomix, a mixture of proinflammatory cytokines. Finally, the shedding of TM and EPCR does not appear to be mediated by increased protein synthesis, but is likely mediated by a matrix metalloproteinase. These findings provide new evidence that the alveolar epithelium can modulate intra-alveolar coagulation through the protein C pathway, and this pathway is likely to be an important determinant of alveolar fibrin deposition.
The endothelial cell surface has previously been thought to be the primary site of protein C activation in vivo (23). Inflammatory stress such as occurs in sepsis leads to a reduction in endothelial activation of protein C and low circulating levels of activated protein C, factors that may contribute to microvascular thrombosis and organ failure in sepsis (24, 25). In cultured endothelial cells the cell surface activity of both TM and EPCR can be down-regulated by inflammatory cytokines such as IL-1 This study provides new information about the mechanisms of shedding of TM and EPCR from the cell surface, an area that is incompletely understood, even in endothelial cells. Prior studies in cultured endothelial cells indicate that EPCR release is a metalloproteolytic process, sensitive to both coagulation factors and inflammatory mediators, but the specific metalloproteinase has not been identified (21). Thrombin or the protease-activated receptors (PAR)1 agonist peptide can enhance EPCR shedding from human umbilical vein endothelial cells via activation of a metalloproteinase, but again the identity of the metalloproteinase is not clear (29). By contrast, shedding of TM is thought to be modulated by neutrophil elastase (30). We identified the mechanisms by which TM and EPCR are shed from alveolar epithelial cells into the conditioned medium independent of neutrophils. Cytomix-induced shedding of TM and EPCR peaked at 12–24 h, suggesting that a rapid induction of a sheddase is not involved. The shedding of both TM and EPCR was blocked by the hydroxamic-based metalloproteinase inhibitors TAPI and GM6001; both inhibitors also partially restored the activation of protein C. This finding suggests that shedding of TM and EPCR was mediated by a metalloproteinase. To further identify the metalloproteinase responsible for TM and EPCR shedding, we studied the effect of TIMP-1, -2, and -3 on EPCR shedding. The nonspecific inhibition pattern for EPCR was not suggestive of a specific metalloproteinase. Inhibition of TM shedding by TIMP 2 and TIMP 3 but not TIMP 1 suggests that MT1-MMP, MT2-MMP, MT3-MMP, MT5-MMP, or MMP-19 is the relevant protease (31).
What are the potential consequences of down-regulation of protein C activation in the setting of acute inflammation in the lung? Recent evidence suggests that APC not only inhibits coagulation but also has anti-inflammatory properties that include suppression of production of cytokines such as TNF- There are some potential limitations of this study. First, many of the studies were done in A549 cells, a cell line that is ultrastructurally similar to the alveolar epithelial type II cell. Although A549 cells are widely used to model the alveolar epithelium, they are derived from a human adenocarcinoma and thus could respond differently than primary type II cells. For this reason we repeated the key experiments in both primary human small airway epithelial cells and primary isolates of human alveolar epithelial type II cells to confirm the findings (Figures 2 and 3). A second possible limitation is that the findings may be pertinent only to cultured alveolar epithelial cells. Although this is a possibility, the finding of high levels of both TM and EPCR in the alveolar space in samples of human pulmonary edema fluid confirms that the intra-alveolar release of these proteins occurs in vivo in ALI/ARDS. The higher levels of TM compared to EPCR in the human pulmonary edema fluid samples may reflect release of TM by additional mechanisms that are not modeled in alveolar epithelial cell culture, such as the previously described shedding of TM by neutrophil elastase (30). A third limitation is that although the findings indicate that both TM and EPCR shedding in response to cytomix is mediated by metalloproteinases, we have not identified the specific metalloproteinases induced by exposure to cytomix. Nevertheless, the information regarding the role of metalloproteolytic cleavage of TM and EPCR from alveolar epithelial cells is new and of likely biological significance. In conclusion, alveolar epithelial cells express the major components of the protein C pathway, including both TM and EPCR, and can activate protein C on the cell surface. Exposure of alveolar epithelial cells to proinflammatory cytokines decreased alveolar epithelial activation of protein C through delayed shedding of TM and EPCR from lung epithelial cells. We conclude that the alveolar epithelium plays an important role in the modulation of intra-alveolar coagulation and inflammation through regulation of the protein C pathway. Down-regulation of activation of protein C by the alveolar epithelial protein C activation may contribute to the pathogenesis of ALI/ARDS and could be a therapeutic target in this disease. Alternatively, the down-regulation of APC may be a well-conserved biological process that allows the host to confine and localize lung injury from infection, such as bacterial pneumonia, with the purpose of preventing systemic dissemination. This interpretation would add further evidence for a pivotal role of the alveolar epithelium in the response to acute lung injury (39–41).
This study was supported by NIH HL70521, HL81332 (Principal Investigator L.W.), P50HL74005, HL51854 (Principal Investigator M.A.M.), and a Biomedical Research Grant from the American Lung Association. Originally Published in Press as DOI: 10.1165/rcmb.2005-0425OC on November 10, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form November 16, 2005 Accepted in final form October 31, 2006
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Abstract
CLINICAL RELEVANCE
P = 0.058 by Mann Whitney U test). (C) Boxplot summary of levels of soluble EPCR in the pulmonary edema fluid from two groups of patients with ALI/ARDS, those with SAPSII of < 45 (n = 18) and those with SAPSII 
