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Published ahead of print on November 20, 2003, doi:10.1164/rccm.200308-1156OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 373-377, (2004)
© 2004 American Thoracic Society

Increased Thrombin Activity after Allergen Challenge

A Potential Link to Airway Remodeling?

Masaki Terada, Elizabeth A. B. Kelly and Nizar N. Jarjour

Pulmonary and Critical Care Medicine Section, Department of Medicine, University of Wisconsin, Madison, Wisconsin

Correspondence and requests for reprints should be addressed to Nizar N. Jarjour, M.D., Pulmonary and Critical Care Medicine Section, 600 Highland Avenue, CSC K4/930 (9988), University of Wisconsin School of Medicine, Madison, WI 53792. E-mail: nnj{at}medicine.wisc.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to its central role in hemostasis, thrombin may play a role in inflammation and remodeling. To investigate the contribution of thrombin to allergic airway inflammation in asthma, we used an enzymatic assay to determine thrombin activity in bronchoalveolar lavage fluid obtained from 19 subjects with atopic asthma before (Day 0) and 48 hours after (Day 2) segmental bronchoprovocation with antigen. Thrombin activity increased from 0 (0, 2.9) on Day 1 to 41.1 (0.3, 75.6) U x 10-3/ml on Day 2 (p = 0.002) and correlated with total protein levels in lavage fluid on Day 2 (r = 0.885, p < 0.001). After antigen challenge, thrombin activity also showed significant correlations with interleukin-5 (r = 0.66, p = 0.002), transforming growth factor ß1 (r = 0.70, p < 0.001), fibronectin (r = 0.85, p < 0.001) and tissue factor (r = 0.55, p = 0.03) levels in lavage fluid. Furthermore, Day 2, but not Day 0 lavage fluid, induced proliferation of human airway fibroblasts. This mitogenic effect was significantly reduced with hirudin, a specific thrombin inhibitor. Taken together, our findings suggest that allergen-driven airway inflammation in asthma is associated with enhanced potential for fibroblast proliferation that is related, at least in part, to increased thrombin activity. We propose that enhanced thrombin activity provides a potential link between allergic inflammation and initiation of airway remodeling.

Key Words: asthma • bronchoalveolar lavage fluid • thrombin • fibroblasts

Airway inflammation is a principle feature of asthma; however, there is increasing evidence that airway remodeling also plays an important part in asthma pathogenesis. Airway remodeling is characterized by thickening of the reticular basement membrane, mucus gland hypertrophy, angiogenesis, deposition of extracellular matrix components, and increased smooth muscle mass (1, 2). Although, structural changes may result in reduced airflow, the role of remodeling in determining asthma severity is controversial (3–6). The relationship between inflammation and remodeling in asthma is not fully established. Although remodeling may be a consequence of repeated injury and persistent inflammation, recent evidence suggests that airway inflammation and remodeling can be parallel, interdependent processes (3, 6–8).

Experimental exposure to allergen results in the accumulation of cells and factors associated with both inflammation and remodeling. Forty-eight hours after segmental bronchoprovocation with antigen (SBP-AG) (9), we have demonstrated both vigorous airway inflammatory responses (10–12) and increased levels of matrix metalloproteinase-9 and fibronectin (12–15), factors with potential roles in airway injury, repair, and remodeling. Similarly, levels of transforming growth factor ß1 (TGF-ß1), a key factor in tissue remodeling and fibrosis (16), are elevated 24 hours after SBP-AG (17). Gizycki and colleagues (18) observed significant increases in "activated fibroblasts" (myofibroblasts) in the airways of subjects with allergic asthma 24 hours after antigen challenge and suggested that repeated allergen exposure may contribute to thickening of the reticular basement membrane and increased smooth muscle mass seen in asthma.

Thrombin is a serine protease that, in addition to its major role in hemostasis (19, 20), participates in a number of processes that mediate inflammation and remodeling in response to tissue injury (21–31). Gabazza and coworkers (32) reported increased thrombin activity in sputum of patients with asthma compared with normal individuals. They further demonstrated that the sputum from subjects with asthma induces proliferation of airway smooth muscle cells (32). Naureckas and colleagues (33) demonstrated that bronchoalveolar lavage (BAL) fluid from patients with asthma had increased mitogenic activity for airway smooth muscle. This mitogenic activity was significantly enhanced 48 hours after SBP-AG (33).

Based on these observations, we hypothesized that antigen challenge induces thrombin activity in the asthmatic airway and that BAL fluid (BALF) from subjects with allergen-challenged asthma is mitogenic for airway fibroblasts. To test this hypothesis, we evaluated thrombin activity in BALF before and 48 hours after SBP-AG in subjects with atopic asthma and determined the effect of BALF on airway fibroblasts proliferation in vitro. Results of this study were previously reported in abstract form (34).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Additional details are provided in the online supplement.

Subject Selection
Nineteen subjects with allergic asthma were studied (Table 1) . BALFs from 6 of the 19 subjects were used in an unrelated study for analysis of soluble interleukin (IL)-5 receptor as previously reported (35). All subjects had FEV1 higher than 70% predicted with reversibility to ß-agonists and/or airway hyperresponsiveness to methacholine (PC20 < 8 mg/ml), and a positive skin-prick test to one or more aeroallergens. No subject smoked or had a respiratory tract infection within 1 month of the study, recent asthma exacerbation, hospitalization, or change in medications for 6 weeks before the study. None of the subjects used systemic or inhaled corticosteroids or leukotriene antagonists in the preceding 6 weeks. Theophylline (for 24–48 hours) and inhaled ß agonists (for 12–24 hours) were withheld before each bronchoscopy. The study was approved by the University of Wisconsin-Madison Center for Health Sciences Human Subjects Committee. All participants gave informed written consent at entry to the study and before each bronchoscopy.


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  		TABLE 1. Subject characteristics

 
SBP and BAL
Bronchoscopy and SBP were performed as described previously (35). Additional details concerning SBP are provided in the online supplement. BAL was performed immediately before (Day 0) and 48 hours after (Day 2) SBP. To control for nonspecific effects of bronchoscopy, six additional subjects with allergy underwent SBP in which saline was instilled into one segment and antigen was instilled in a separate segment (13). BAL was performed in each segment immediately and 48 hours after SBP.

Thrombin Assay
Thrombin activity was measured by enzymatic assay, using the chromogenic substrate, H-D-hexahydrotyrosol-alanyl-arginine-para-nitroanilide diacetate (American Diagnostica, Greenwich, CT) (36, 37) (see the online supplement for details). In brief, BALF or serial dilutions of known concentrations (250 x 10-3 to 3 x 10-3 National Institutes of Health U/ml) of {alpha}-thrombin (American Diagnostica) were added to the substrate and optical density was measured over a 2-hour time period. Data are presented as National Institutes of Health U/ml of 1 x BALF.

ELISAs
A sandwich-type ELISA was used to measure cytokines, extracellular matrix, and growth factors in BALF as described previously (11). The tissue factor was measured using a commercial kit (IMUBIND Tissue Factor ELISA Kit; American Diagnostica) (see online supplement for additional details).

Fibroblast Proliferation Assay
Details of fibroblast culture and proliferation assays are provided in the online supplement. In brief, bronchial fibroblast cultures (10,000 cells/well) were stimulated for 24 hours with BALF (diluted 1:20 in serum-free medium) in the presence or absence of 0.5 U/ml of hirudin (Calbiochem, San Diego, CA). Fibroblast proliferation was evaluated by incorporation of bromodeoxyuridine.

Statistical Analysis
Wilcoxon's signed rank test (or a paired Student's t test for normally distributed data) was used to compare BALF cells and factors obtained before and after antigen challenge (38). For fibroblast proliferation, a paired Student's t test was used to compare cells between treatments. Correlations were tested with Spearman's rank order correlation. For all comparisons, statistical significance was accepted as p values less than 0.05. Statistical analysis was performed using SigmaStat 2.03 (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Thrombin Activity in BAL Fluid before and 48 hours after SBP-AG
Thrombin activity was significantly increased after antigen challenge, from undetectable levels (median and 25% interquartile were not detectable, 75% interquartile was 31 x 10-3 U/ml) on Day 0 to 50 x 10-3 (3 x 10-3, 85 x 10-3) U/ml, (median with 25 and 75% interquartile range) on Day 2 (p = 0.002, Figure 1) . To establish that the changes were due to antigen challenge, six additional subjects were evaluated after challenge with saline in one segment and antigen in a second segment. Although there was only a modest (fourfold) increase in thrombin activity in the saline-challenged segment, there was a striking increase (20-fold) after antigen challenge.



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  		Figure 1. Comparison of thrombin activity in bronchoalveolar lavage fluid (BALF) before (Day 0) and 48 hours after (Day 2) segmental bronchoprovocation in subjects with atopic asthma (n = 19). Horizontal lines represent mean values.

 
Cellular Profiles and Cytokine, Growth Factor, and Extracellular Matrix Levels in BAL Fluid before and 48 hours after SBP-AG
There was a significant increase in total cells (p < 0.001) and in the percentage of eosinophils (p < 0.001) and neutrophils (p < 0.001) in BAL 48 hours after SBP-AG (Table 2) . As shown in Table 3 , levels of total BAL protein (p < 0.001), IL-5 (p < 0.001), IL-8 (p = 0.008), IL-13 (p < 0.001), TGF-ß1 (p = 0.003), and fibronectin (p < 0.001) on Day 2 were significantly higher than those on Day 0. The change in tissue factor levels was not statistically significant.


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  		TABLE 2. Cellular profiles and total protein in bronchoalveolar lavage fluid before (day 0) and 48 h (day 2) after segmental bronchoprovocation

 

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  		TABLE 3. Factors in bronchoalveolar lavage fluid before (day 0) and 48 h (day 2) after segmental bronchoprovocation

 
Thrombin activity (U/ml) in BALF on Day 2 correlated with the levels of TGF-ß1 (r = 0.70, p < 0.001, Figure 2A) and fibronectin (r = 0.85, p < 0.001, Figure 2B), factors that may play a role in airway remodeling. In addition, thrombin activity correlated with markers of airway inflammation, including total protein levels (r = 0.89, p < 0.001), concentration (cells/ml) of total cells (r = 0.86, p < 0.001), the relative percentage of eosinophils (r = 0.60, p = 0.007), and levels of the eosinophil-related cytokine, IL-5 (r = 0.66, p = 0.002, Figure 2C). There was also a modest correlation between thrombin activity and the concentration of soluble tissue factor in BALF on Day 2 (r = 0.55, p = 0.03, Figure 2D). In contrast, there was no relationship between BALF levels of thrombin and levels of IL-8 or IL-13.



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  		Figure 2. Correlation between thrombin activity and transforming growth factor ß1 (TGF-ß1) (A), fibronectin (B), interleukin (IL)-5 (C), and tissue factor (D) in BALF 48 hours after segmental bronchoprovocation.

 
Effect of BAL Fluid on Fibroblast Proliferation
To determine the potential role of thrombin in BALF, we measured the mitogenic activity of BALF from subjects with the highest thrombin activity on Day 2 (Figure 3) . As a positive control, bronchial fibroblasts were cultured for 24 hours with thrombin in the presence or absence of the specific thrombin inhibitor, hirudin. Thrombin induced significant proliferation of fibroblasts, which was inhibited by hirudin. BALF obtained after antigen challenge (Day 2) also induced fibroblast proliferation, which was nearly completely inhibited by hirudin. BALF from Day 0 had no effect on fibroblast proliferation.



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  		Figure 3. Effect of BALF obtained before and 48 hours after segmental bronchoprovocation on fibroblast proliferation. Normal human airway fibroblasts were stimulated for 24 hours with (A) serum-free medium (none) or 0.1 U/ml of thrombin or (B) BALF obtained before (Day 0) and 48 hours after (Day 2) antigen challenge in the presence (hatched bars) or absence (open bars) of 0.5 U/ml of hirudin. Cellular proliferation was evaluated by bromodeoxyuridine assay. Data are presented as means ± SEM of optical density (OD) at 450 nm. (*p values less than 0.05 versus none, {dagger}p values less than 0.05 versus with hirudin, §p values less than 0.05 versus BALF on Day 0).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated increased thrombin activity in BALF from patients with asthma challenged with allergen. Thrombin activity in BALF correlated with leukocyte influx and with levels of IL-5, fibronectin, TGF-ß1, and to a lesser extent, soluble tissue factor. Furthermore, BALF obtained 48 hours after SBP-AG was mitogenic for airway fibroblasts, and this mitogenic activity was inhibited by the thrombin-specific inhibitor, hirudin. Enhanced thrombin activity is thus associated with allergic airway inflammation and may, by promoting the proliferation of bronchial fibroblasts, participate in airway remodeling.

Our data confirm and extend previous observations by Gabazza and coworkers and Naureckas and colleagues showing that sputum (32) and BALF (33) obtained from patients with asthma induces proliferation of airway smooth muscle cells. Gabazza and colleagues demonstrated elevated baseline levels of thrombin activity in sputum from asthma subjects compared with sputum from normal, healthy subjects (32). Naureckas and coworkers (33) described an airway smooth muscle cell mitogenic factor in BALF that increased 48 hours after airway antigen challenge. The mitogenic factor described in their study was found to have a molecular weight greater than 10 kD but was not further identified. Thrombin, with a molecular weight of 37 kD, is a potential candidate for the mitogenic factor described in that study. In addition to asthma, there is growing evidence that thrombin is released into the airway during chronic airway inflammation and injury and that it has the capacity to participate in fibroblast proliferation. Thrombin activity has been described in BALF from premature infants with chronic lung disease (39), patients with systemic sclerosis (40), and experimental radiation pneumonitis in rats (41). In each case, BALF induced fibroblast proliferation, which was attenuated with thrombin inhibitors.

As we have previously demonstrated, segmental allergen exposure induces a vigorous inflammatory response in the asthmatic airway (10, 11). In the present study, thrombin activity was related to the intensity of airway inflammation, correlating with total cell numbers, relative percentage of eosinophils, and total protein levels at Day 2. Thrombin activity was also correlated at Day 2 with levels of IL-5, a key cytokine for eosinophilic inflammation in the asthmatic airway (11). Although we cannot conclude a causal relationship between thrombin levels and inflammation, previous in vitro and in vivo studies have shown that thrombin is involved in endothelial permeability (42, 43) and leukocyte chemotaxis (44–47). Thus, thrombin might play a role in increasing permeability and cell migration processes in allergic airway inflammation.

Because fibronectin increases leukocyte chemotaxis, alters leukocyte phenotypes (48), and induces fibroblast proliferation (49), it is believed to play a role in the pathogenesis of asthma (50, 51). A number of factors, including thrombin and TGF-ß1 are potent inducers of fibronectin generation by fibroblasts (31). As we have shown previously (14), SBP-AG resulted in increased levels of airway fibronectin; moreover, in this study, thrombin activity was positively correlated with fibronectin levels at Day 2. Likewise, we, and others (17), have demonstrated elevated levels of TGF-ß1, a factor involved in tissue regeneration, remodeling, and fibrosis (16), in BALF after SBP-AG, and we found a positive correlation between levels of thrombin and TGF-ß1 in Day 2 BALF. Increased thrombin activity in the airway may participate, with fibronectin and TGF-ß1, in inflammatory and remodeling processes in the asthmatic airway.

Contact with tissue factor triggers the coagulation cascade, resulting in the conversion of prothrombin to active thrombin (52). Levels of both thrombin and soluble tissue factor are elevated in the sputum of patients with stable asthma compared with sputum from healthy control subjects (32). Our present data showed that although tissue factor was not significantly increased after antigen challenge, there was a modest, but significant, correlation between tissue factor and thrombin activity in BALF on Day 2 after SBP-AG. It is likely that only a portion of the tissue factor in the airway was detected by analysis of BALF. An increase in the expression of tissue factor on the surface of airway cells would not have been detected by our ELISA assay.

We recognize some limitations in this study. First, it may be argued that SBP-AG is an intense stimulus and the resultant increase in mitogenic activity of BALF on ex vivo–stimulated fibroblasts may not reflect ongoing events in vivo. Gizycki and colleagues (18), however, performed aerosol allergen challenge and observed increases in the number of activated fibroblasts in biopsied tissue, suggesting that antigen challenge may indeed induce proliferation in vivo. Second, the substrate used in the enzymatic assay may not have absolute specificity for thrombin. The biological assay, however, confirmed the presence of thrombin in BALF on Day 2 because hirudin is a specific inhibitor of thrombin activity. Although other factors may have been detected by the enzymatic assay, our findings suggest that thrombin is a key contributor to fibroblast proliferation. The fibroblast proliferation assays were performed in the subset of BALF with the highest thrombin enzymatic activity, raising the question of physiologic relevance of thrombin levels in BALF. However, it is important to keep in mind that BALF likely represents 100-fold dilution of the epithelial lining fluid. Therefore, one would expect thrombin activity to be severalfold greater in vivo, and at these levels, thrombin is known to be an effective fibroblast mitogen.

In summary, we have demonstrated an association between allergen-driven inflammation and increased thrombin activity in the BALF of subjects with asthma. Enhanced thrombin activity in BALF was correlated with leukocyte influx and with levels of IL-5, TGF-ß1, fibronectin, and tissue factor. Moreover, BALF from allergen-stimulated airways was mitogenic for airway fibroblasts. These findings suggest that thrombin activity induced by allergen exposure may play a role in asthma pathogenesis by participating in airway inflammation and initiating remodeling.


   Acknowledgments
 
The authors thank Sarah Panzer and Amy Neeno-Eckwall for assistance with processing BALF and establishing the fibroblast cell line; Keith Meyer M.D., Ann Dodge R.N., Andrea Tweedie R.N., and Mary Jo Jackson R.N. for assistance with patient recruitment, screening, and bronchoscopies; Deane Mosher, M.D., Robert Schilling, M.D., and William Busse, M.D., for their valuable suggestions; and Jacqueline Houtman, Ph.D., for assistance with manuscript preparation.


   FOOTNOTES
 
Supported by National Institutes of Health grants HL64066, K24-AI01704, and MOI RR-03186 from the National Center for Research Resources.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: M.T. has no declared conflict of interest; E.A.B.K. has no declared conflict of interest; N.N.J. has no declared conflict of interest.

Received in original form August 19, 2003; accepted in final form November 18, 2003


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