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PEDIATRICS Vol. 112 No. 3 September 2003, pp. 570-577

Pulmonary Trypsin-2 in the Development of Bronchopulmonary Dysplasia in Preterm Infants

Katariina Cederqvist, MD*, Caj Haglund, MD, PhD{ddagger}, Päivi Heikkilä, MD, PhD§, Timo Sorsa, DDS, PhD||, Taina Tervahartiala, DDS||, Ulf-Håkan Stenman, MD, PhD and Sture Andersson, MD, PhD*,#

* Hospital for Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland
{ddagger} Departments of Surgery
§ Pathology
|| Oral and Maxillofacial Diseases and Institute of Dentistry
Clinical Chemistry
# Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Objectives. In the preterm infant, lung injury can lead to irreversible tissue destruction and abnormal lung development. We examined whether pulmonary trypsin, a potent matrix-degrading serine proteinase and proteinase-cascade activator, is associated with the development of bronchopulmonary dysplasia (BPD) in preterm infants.

Methods. Samples of tracheal aspirate fluid were collected from 32 intubated preterm infants during their first 2 postnatal weeks. The presence and molecular forms of trypsin in tracheal aspirate fluid samples were analyzed by zymography and Western blotting. The concentrations of trypsinogen-1 and -2 and tumor-associated trypsin inhibitor were measured by immunofluorometry. For examining the expression of trypsin-2 in lung tissue, immunohistochemistry was performed on autopsy specimens of fetuses, of preterm infants who died from respiratory distress syndrome or BPD, and of term infants without lung injury.

Results. In infants who subsequently developed BPD (n = 18), we detected significantly higher concentrations of trypsinogen-2 during postnatal days 5 to 10 compared with those who survived without it. There was no difference in trypsinogen-1 concentrations. Tumor-associated trypsin inhibitor concentrations were significantly lower in infants who needed mechanical ventilation for >1 week. Immunohistochemistry demonstrated that trypsin-2 was predominantly expressed in bronchial and bronchiolar epithelium. In 2 preterm infants who died from prolonged respiratory distress syndrome, trypsin-2 was also expressed in vascular endothelium.

Conclusions. The levels of trypsinogen-2 are higher during postnatal days 5 to 10 in infants who subsequently develop BPD. The results suggest that high levels of pulmonary trypsin-2 may be associated with the development of BPD. This raises the possibility that therapy with exogenous proteinase inhibitors might prevent the development of BPD in preterm infants with respiratory distress.

Key Words: trypsin • preterm infants • respiratory distress syndrome • bronchopulmonary dysplasia

Abbreviations: ECM, extracellular matrix • BM, basement membrane • MMP, matrix metalloproteinase • PSTI, pancreatic secretory trypsin inhibitor • TATI, tumor-associated trypsin inhibitor • BPD, bronchopulmonary dysplasia • RDS, respiratory distress syndrome • TAF, tracheal aspirate fluid • SD, standard deviation • GA, gestational age • BW, birth weight • SC, secretory component • IgA, immunoglobulin A • L/S, lecithin/sphingomyelin • aAPO2, arterial to alveolar oxygen tension ratio

Trypsin is a serine proteinase secreted as a zymogen (trypsinogen) in pancreatic fluid and activated by enterokinase in the intestine, where it plays an important role as a food-digestive enzyme. In humans, there are 4 known trypsin isoenzymes, of which trypsin-1, -2, and -3 are expressed in the pancreas.1,2 Trypsin-1 and -2 are also expressed in several types of human cancer cells3 and in normal human epithelial cells of the esophagus, kidney, and lung, as well as leukocytes in the spleen, and in vascular endothelial cells.4–6 Trypsin-2 can directly attack various extracellular matrix (ECM) and basement membrane (BM) proteins7; in addition, at very low concentrations, it efficiently activates latent forms of matrix metalloproteinases (MMPs), thus owing potential to initiate a broad-spectrum proteinase cascade eventually leading to tissue destruction.8 Trypsin and trypsin-like proteinases, such as tryptase, may also play a role in inflammatory and fibroproliferative processes.9,10

The activity of trypsin is controlled by several inhibitors, such as {alpha}1-antitrypsin, {alpha}2-macroglobulin, and pancreatic secretory trypsin inhibitor (PSTI), which is also called tumor-associated trypsin inhibitor (TATI).11,12 PSTI, or TATI, is a highly specific inhibitor of trypsin originally found in pancreas, but it is now known to be widely expressed in mucus-producing cells of gastrointestinal tract, in kidney, and in lung.13 TATI is expressed in several cancers together with trypsinogen.14 The major physiologic roles of PSTI, or TATI, are thought to be to prevent premature activation of trypsinogen in pancreas and to protect mucosal cells of gastrointestinal tract from proteolytic attack.15,16 It may also play a role in epithelial repair.13 In tumors, TATI is thought to protect the tumor from destruction by trypsin.8,14

Despite the use of exogenous surfactant and the advances in neonatal intensive care, bronchopulmonary dysplasia (BPD) remains a common chronic disease of very low birth weight preterm infants.17 Lung immaturity, oxidative stress, mechanical ventilation, and pulmonary inflammation are considered to be major factors involved in the pathogenesis of BPD.18–21 In preterm infants with respiratory distress syndrome (RDS), pulmonary injury caused by oxygen toxicity and mechanical ventilation is thought to induce an inflammatory reaction in the airways and interstitium of the immature lungs. This pulmonary inflammation is associated with increased alveolar capillary permeability, which is at least partly caused by disruption of BMs.22,23 Destruction of ECM and BM components may affect alveolar development, because besides structural roles, ECM proteins regulate cell growth and differentiation. As a potent matrix-degrading proteinase and MMP activator, trypsin-2 might play an important role in the damage of ECM and BM in preterm lung injury.7,8 Disorganized lung architecture, impaired alveolarization, and interstitial proliferation are prominent pathologic findings in BPD, but the pathogenetic mechanisms that lead to tissue destruction and impairment of lung development are still not clear.

We hypothesized that trypsin, especially when insufficiently counteracted by its specific locally produced inhibitor TATI, can play a role in the development of BPD in preterm infants. Therefore, the presence and molecular forms of trypsin were studied by gelatin zymography and Western blotting, and the concentrations of trypsinogen-1 and -2 and TATI were measured in tracheal aspirate fluid (TAF) samples of preterm infants with respiratory distress during the first 2 postnatal weeks. The localization of pulmonary trypsin-2 was examined by immunohistochemistry in autopsy specimens of fetuses, of infants with RDS or BPD, and of full-term infants without primary lung injury.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
All studies were done with the approval of the Ethics Committee of the Hospital for Children and Adolescents, University Central Hospital, Helsinki.

Patients
Preterm infants (n = 32) who had respiratory distress and were admitted to the neonatal intensive care unit of the Hospital for Children and Adolescents, University of Helsinki, were enrolled between December 1993 and October 1997. All were intubated at birth because of failure to establish spontaneous ventilation. Infants with major anomalies were excluded.

To the mothers, antenatal glucocorticoids were given as 12 mg of betamethasone twice with a 12-hour interval. Chorioamnionitis was diagnosed on the basis of clinical signs with leukocytosis (>15 x 109 cells/L) and increased concentration of C-reactive protein (>50 mg/L).

All infants received ampicillin 200 mg/kg/d and nethilmicin 6 mg/kg/d from the first day of life; for 7 infants, ampicillin was changed to vancomycin 15 mg/kg/d during the first postnatal week because of clinical signs of septic infection; in 1 of them, diagnosis was verified by positive blood culture. Treatment with indomethacin for patent ductus arteriosus was given as 4 doses of 0.1 mg/kg at 12-hour intervals. For facilitating weaning from mechanical ventilation, treatment with dexamethasone was given at a dose of 0.5 mg/kg/d for 3 days, followed by 0.25 mg/kg/d for 3 to 5 days and 0.125 mg/kg/d for 3 to 5 days, starting at a mean (±standard deviation [SD]) age of 13 ± 7 days (range: 3–33 days). For 3 of these infants, dexamethasone was started during the first postnatal week on days 3, 6, and 7.

Development of BPD was defined as the need for supplemental oxygen at the age of 36 gestational weeks, in association with chest radiographic findings typical for BPD.24 One infant (GA 27.1 weeks) died from severe BPD at 6 months of age. Patient characteristics are given in Table 1.


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  		TABLE 1. Patient Characteristics According to Development of BPD

 
Autopsied Subjects
A total of 8 fetuses that were aborted between August 1998 and October 1999 and 19 infants who died between January 1991 and October 1998 in the University Central Hospital, Helsinki, were studied. Of the 8 fetuses (age at death: 14–29 weeks; weight: 30–1240 g), 7 were aborted because of major extrapulmonary anomalies and 1 because of placental ablation. Only fetuses with microscopically and macroscopically normal lungs were included. The studied 19 infants were as follows: preterm infants who died of RDS at the age of 0 to 3 days (n = 6; gestational age [GA]: 23.0–27.7 weeks; birth weight [BW]: 310–840 g), preterm infants who died of RDS at the age of 9 to 16 days (n = 3; GA: 24.6–29.9 weeks; BW: 500–810 g), preterm infants who died of BPD at the age of 75 to 285 days (n = 5; GA: 25.1–29.0 weeks; BW: 670-1070 g), and term infants who had normal lung histology and died from hypoplastic left heart syndrome or Ebstein anomaly at the age of 1 to 3 days (n = 5; GA: 38.3–41.0 weeks; BW: 2900–4270 g). None of the infants presented with any lung anomalies or pneumonia at the time of death. Autopsies were performed within 2 days after death. The lung samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and stored at room temperature.

Sampling of TAF
TAF samples were collected once daily by standardized routine tracheal lavage as previously described.20 The tubes were stored at –20°C until analysis.

Gelatin Zymography
For analysis of gelatinolytic activity by zymography, TAF samples were run on 1.5-mm-thick 8% to 10% sodium dodecyl sulfate-polyacrylamide gels impregnated with 1 mg/mL gelatin (Sigma, St Louis, MO) as described previously.25

Western Immunoblotting
The molecular forms of the trypsin isoenzymes in TAF samples were analyzed by Western blot analysis using a specific polyclonal rabbit antiserum.26 After electrophoresis, the proteins in the gel were electrotransferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Richmond, CA). After the unoccupied sites were blocked with gelatin, the membrane first reacted with the primary antibody (1:500) and then with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (1:1000; Sigma). The immunoreactive proteins were visualized with Nitro Blue Tetrazolium (Sigma) and 5-bromo-4-chloro-3-indolyl-phosphate (Sigma) solution.

Immunofluorometric Assays
The concentrations of trypsinogen-1 and -2 and TATI in TAF were determined by specific time-resolved immunofluorometric assays as described previously.27,28 The detection limit was 0.1 µg/L for trypsinogen-1, 0.3 µg/L for trypsinogen-2, and 0.2 µg/L for TATI. The inter- and intra-assay coefficients of variation were 10% to 15% for trypsinogen-1, 10% to 12% for trypsinogen-2, and 13% to 14% for TATI.27,28

Secretory Component of Immunoglobulin A
The concentrations of trypsinogen-1 and -2 and TATI in TAF were related to concentrations of secretory component (SC) of immunoglobulin A (IgA) as the reference protein by direct enzyme-linked immunosorbent assay as previously described.29,30

Lecithin/Sphingomyelin Ratio and Phosphatidyl Glycerol
For determining surfactant maturity, TAF samples were collected within 3 hours after birth before treatment with surfactant. The samples were analyzed for lecithin/sphingomyelin (L/S) ratio and phosphatidyl glycerol by thin-layer chromatography as a part of our clinical routine.

Immunohistochemistry
Trypsin-2 immunoreactivity was visualized with monoclonal anti-trypsin-2 antibody (8F7).27 The sections were deparaffinized in xylene and rehydrated through graded concentrations of ethanol to distilled water and processed in a microwave oven. This procedure of antigen retrieval was found to give better staining than did pretreatment with pepsin or no pretreatment. The sections were then treated with 0.5% hydrogen peroxide in methanol for 30 minutes to quench endogenous peroxidase activity and blocked with normal horse serum (1:20) for 15 minutes to reduce nonspecific binding. Primary antibody to trypsin-2 (diluted 1:1000) was added, and the sections were incubated overnight at room temperature. Bound antibody was visualized by the avidin-biotin complex immunoperoxidase technique (Elite ABC Kit, Vectastain; Vector Laboratories, Burlingame, CA) following the manufacturer’s instructions. The sections were incubated with the biotinylated second layer antibody and the peroxidase-labeled avidin-biotin complex for 30 minutes each. The peroxidase staining was visualized with 3-amino-9-ethyl-carbazole (A-5754;Sigma), 0.2 mg/mL in 0.05 M acetate buffer containing 0.03% H2O2, pH 5.0 for 15 minutes at room temperature. Counterstaining was performed with Mayer’s hematoxylin solution. Sections that were treated with nonimmune mouse serum or phosphate-buffered saline served as negative controls. Pancreatic specimens were used as positive controls.

Statistical Analysis
Comparisons between unpaired data were performed with Mann-Whitney U test or Fisher’s exact text. Logarithmic transformation of the data were performed for linear regression analysis used for continuous variables. P < .05 was considered statistically significant. Patient data are given as mean ± SD, and results are given as medians and ranges and interquartiles. All calculations were done with StatView 5.0.1 (Abacus Concepts Inc, Berkeley, CA).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
A total of 249 TAF samples were collected from the 32 infants during their first 2 postnatal weeks. The mean amount of samples for each infant was 7.3 ± 2.7 (SD).

Characterization of Trypsin in TAF
In TAF samples, Western blotting showed the presence of 25 to 28 kDa, 40 kDa, 55 kDa, and 100 kDa immunoreactive species of trypsin (Fig 1). Of these, the 25- to 28-kDa major immunoreactive trypsin species corresponded to the 25- to 28-kDa gelatinolytic enzyme demonstrated by zymography, and the 40-kDa, 55-kDa, and 100-kDa relatively faint trypsin immunoreactive species probably represented complexes between trypsin and inhibitors (Fig 1). The 25-kDa band corresponds to trypsin-1, and the 28-kDa band corresponds to trypsin-2.26 The 90-kDa gelatinolytic proteinase in TAF detected by zymography has been identified by Western blotting to be gelatinase B, also called MMP-9.30


Figure 1
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  		Fig 1. Gelatin zymogram (lane 1) and Western immunoblotting (lane 2) for trypsin-1 and -2 of TAF from a preterm infant with respiratory distress. The 25- to 28-kDa gelatinolytic bands detected by zymography correspond to the trypsin bands detected by immunoblotting. The positions of molecular weight standards are indicated by kDa. *High molecular weight complexed forms of trypsin.

 
Concentrations of Trypsinogen-1 and -2 and TATI in TAF
The concentrations of trypsinogen-1 and -2 were above the detection limit of the assay in 86% and 96% of the samples, respectively. The concentrations of TATI were measured in a subset of 197 TAF samples, and they were above the detection limit in 97% of these samples.

During weeks 1 and 2, the median concentrations of trypsinogen-1 were 1.6 ng/SC unit (range: 0.0–95.0) and 0.9 ng/SC unit (range: 0.0–17.2), and of trypsinogen-2 14.5 ng/SC unit (range: 0.0–235.4) and 17.3 ng/SC unit (range: 0.0–266.6). A positive correlation existed between the concentrations of trypsinogen-1 and -2 during week 1 (r = 0.49) and week 2 (r = 0.45; both P < .0001). The median concentration of TATI was 35.1 ng/SC unit (range: 3.9–925.7) during week 1 and 21.0 ng/SC unit (range: 2.6–342.1) during week 2. A positive correlation existed between the concentrations of TATI and trypsinogen-1 (week 1: r = 0.41, P < .001; week 2: r = 0.44, P < .0001) and of TATI and trypsinogen-2 (week 1: r = 0.34, P < .01; week 2: r = 0.50 P < .0001).

During week 1, trypsinogen-1 showed a positive correlation with gestational age (r = 0.36, P < .001). The concentration of trypsinogen-1 did not correlate with proteinuric preeclampsia, antenatal glucocorticoid treatment, BW, severity of acute respiratory distress, or duration of mechanical ventilation (data not shown).

A weak negative correlation was observed between trypsinogen-2 concentration during week 1 and BW (r = –0.26, P = .005), whereas there was no correlation with gestational age during week 1 or 2 (data not shown). No association existed between trypsinogen-2 and antenatal glucocorticoid treatment. Trypsinogen-2 was significantly higher during weeks 1 and 2 in infants who were born to mothers with proteinuric preeclampsia than in those who were born to mothers with chorioamnionitis or premature rupture of the membranes or in those who were born to mothers without these complications (Fig 2). The infants who were born to mothers with proteinuric preeclampsia (n = 11) were of a higher GA (mean [±SD]: 28.6 ± 1.8 weeks vs 26.6 ± 1.8 weeks, respectively; P = .003) but had more severe acute respiratory distress as indicated by a lower initial arterial to alveolar oxygen tension ratio (aAPO2; mean [±SD] 0.15 ± 0.07 vs 0.33 ± 0.22; P = .02). A negative correlation existed between trypsinogen-2 during weeks 1 and 2 and surfactant maturity, as measured as L/S ratio in TAF (week 1: r = –0.36, P = .001; week 2: r = –0.66, P < .0001). Furthermore, the presence of phosphatidyl glycerol in TAF was associated with lower trypsinogen-2 levels during weeks 1 and 2 (Table 2). Trypsinogen-2 was higher in infants with an initial aAPO2 ≤0.22 and in those who needed treatment with surfactant (Table 2). The concentration of trypsinogen-2 during week 1 showed a positive correlation with duration of mechanical ventilation (r = 0.30, P = .001).


Figure 2
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  		Fig 2. Box plot of concentrations of trypsinogen-2 in TAF during weeks 1 and 2 in infants who were born to mothers with proteinuric preeclampsia, with premature rupture of the membranes (PROM) or chorioamnionitis, or without these complications. Box denotes the 25th, 50th, and 75th percentiles; whiskers represent the 10th and 90th percentiles. Trypsinogen-2 concentrations corrected for dilution by division with concentrations of SC of IgA. Results are expressed as ng/SC unit. The number of samples analyzed is shown in parentheses. *P < .05; **P < .0001.

 
A weak positive correlation appeared between TATI concentrations during weeks 1 and 2 and BW (week 1: r = 0.27, P = .008; week 2: r = 0.38, P = .0002). No correlation existed between TATI and proteinuric preeclampsia or severity of acute respiratory distress (data not shown), but the concentrations during week 1 were significantly lower in infants who needed mechanical ventilation for >1 week (median [range] 27.2 ng/SC unit [3.9–925.7] vs 61.8 ng/SC unit [18.4–534.0]; P < .0001).

Trypsinogen-1 and -2 Concentrations in TAF and Subsequent Development of BPD
Figure 3 illustrates the concentrations of trypsinogen-2 through the study period of first 2 postnatal weeks in infants who subsequently developed BPD and in those who did not. The concentration was significantly higher in the BPD group on days 5 to 10 (Fig 3). During weeks 1 and 2, the ratio of trypsinogen-2 to TATI was higher in infants who subsequently developed BPD than in those who survived without it (Fig 4). There were no differences in trypsinogen-1 concentrations between the 2 groups (data not shown).


Figure 3
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  		Fig 3. Box plot of concentrations of trypsinogen-2 in TAF during the first 2 postnatal weeks postnatal age divided into 2 day time periods in infants who subsequently developed BPD and in those who survived without BPD. Box denotes the 25th, 50th, and 75th percentiles; whiskers represent the 10th and 90th percentiles. Trypsinogen-2 concentrations corrected for dilution by division with concentrations of SC of IgA. Results are expressed as ng/SC unit. The number of samples analyzed is shown in parentheses. *P < .05.

 

Figure 4
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  		Fig 4. Box plot of ratios of trypsinogen-2 to TATI in TAF during weeks 1 and 2 in infants who subsequently developed BPD and in those who survived without BPD. Box denotes the 25th, 50th, and 75th percentiles; whiskers represent the 10th and 90th percentiles. The number of samples analyzed is shown in parentheses.

 
Trypsin-2 Immunohistochemistry
Immunohistochemical staining of lung sections from fetuses revealed a weak positive staining reaction in bronchial and bronchiolar epithelial cells in 2 cases (Fig 5A). However, 6 cases were totally negative. Lung sections from preterm infants who died of RDS at the age of 0 to 3 days were mainly negative in immunostaining. In 3 of these infants, a faint positive staining was detected in bronchial epithelial cells. In addition, intra-alveolar fluid and hyaline membranes were slightly positive (Fig 5B). In contrast, in preterm infants who died of severe RDS at the age of 9 to 16 days, bronchial and bronchiolar epithelial cells were strongly positive. In 2 of these infants, vascular endothelial cells also showed positive staining, and in 1 intense immunostaining was detected also in type II-like pneumocytes. Intra-alveolar fluid and hyaline membranes were positive similarly to that seen in preterm infants who died of RDS at the age of 0 to 3 days (Fig 5C and D). In infants who died of BPD, bronchial and bronchiolar epithelial cells, as well as bronchial metaplastic stratified squamous epithelium, predominantly showed weak positive staining. In only 1 infant, intense positive staining was detected in both bronchial and bronchiolar epithelium and in type II-like pneumocytes (Fig 5E). Lung sections from term infants who had normal lung histology and died at the age of 1 to 3 days demonstrated faint staining of bronchial and bronchiolar epithelial cells. In 1 infant, moderate positive staining was also detected in type II-like pneumocytes (Fig 5F).


Figure 5
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  		Fig 5. Immunohistochemical staining (original magnification: x130) for trypsin-2 in lung. A, Fetus (age at death: 22 weeks). B, Preterm infant who died of RDS at the age of 3 days (GA: 25.7 weeks). C, Preterm infant who died of RDS at the age of 9 days (GA: 29.9 weeks). D, Preterm infant who died of RDS at the age of 11 days (GA: 24.6 weeks). E, Preterm infant who died of BPD (GA: 28.9 weeks; age at death: 83 days). F, Term infant who died of hypoplastic left heart syndrome (GA: 38.3 weeks; age at death: 1 day). Asterisk, artery; arrow, capillary; br, bronchus; al, alveolus or airspace.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
We show here that in preterm infants, the development of BPD is associated with high pulmonary concentrations of trypsinogen-2 during the early postnatal period. In addition, the ratio of trypsinogen-2 to its specific inhibitor TATI is higher in infants who subsequently develop BPD.

Immunohistochemical analysis showed that pulmonary trypsin-2 was predominantly expressed in bronchial and bronchiolar epithelial cells. It is interesting that positive staining could not be detected in inflammatory cells. The strongest expression was detected in lungs of preterm infants who died from RDS at the age of 9 to 16 days. In these, intense expression of trypsin-2 was detected in bronchial and bronchiolar epithelium, and in 2 infants, also in vascular endothelium. Previously, expression of trypsin-2 in vascular endothelium has been detected in patients with disseminated intravascular coagulation and also around gastric tumors, which has been suggested to facilitate tumor angiogenesis through BM degradation.5 Accordingly, in preterm infants with prolonged RDS, trypsin-2 in vascular endothelium may play a role in the degradation of the alveocapillary barrier. In the lungs of term infants with normal histology, trypsin-2 was expressed at low levels in bronchial and bronchiolar epithelial cells, a finding in accordance with those of earlier studies showing expression of trypsin in adult human bronchial epithelial cells and supporting the hypothesis that trypsin may also participate in normal cellular functions.4,6

We found no association between gestational age and trypsinogen-2 levels; however, a negative correlation existed between trypsin-2 levels and the functional maturity of the alveolar type II cells defined on the basis of the L/S ratio and the presence of phosphatidyl glycerol in TAF. In addition, we detected higher levels of trypsin-2 in infants who were born to mothers with preeclampsia. These infants presented with an even more difficult early lung disease, as indicated by a lower initial aAPO2. Hypertensive pregnancy has been shown to be associated with lower fetal lung maturity and with low concentrations of surfactant protein A in the preterm lung31,32 and, in addition, with the development of BPD.32 Thus, trypsin-2 may contribute to the development of BPD in these infants.

In the lungs, trypsin-2 may contribute to tissue injury through several mechanisms. Trypsin-2 is a potent matrix-degrading proteinase: it directly degrades various components of the ECM and BMs and efficiently activates latent MMPs at very low concentrations.7,8 Activation of MMP cascade has been shown in lungs of preterm infants.30 Increased pulmonary microvascular permeability is a characteristic feature in RDS33 and is also associated with the development of BPD.23 Disruption of BMs and remodeling of the ECM by proteolytic enzymes may contribute to the changes in the alveocapillary barrier. Breakdown of glycosaminoglycans on the vascular endothelial BM and interstitium has been demonstrated in the lungs of infants who died of RDS.34 In experimental pancreatitis-associated lung injury, intravenous infusion of trypsin or trypsinogen into healthy rats induces an acute dose-dependent pulmonary injury characterized by perivascular edema and hemorrhage.35 Thus, increased trypsin load in the preterm lung, especially in association with low TATI levels, could represent local imbalance sufficient to cause proteolytic injury.

Trypsin can cleave complement component C5, thus generating the anaphylatoxin C5a, which has chemotactic and neutrophil-activating characteristics.36 In lungs of preterm infants with RDS, high chemotactic activity of TAF, high levels of C5a, and recruitment of neutrophils are associated with the development of BPD.22,23

Both pancreatic trypsin and extrapancreatic trypsin-2 are potent activators of proteinase-activated receptor-2 (PAR-2).9,37,38 PAR-2 is a member of a family of G protein-coupled receptors cleaved by serine proteases within the extracellular N-terminal domain, allowing the new N-terminus to bind and activate the receptors themselves.37 Recent studies suggest that activation of the PAR-2 receptor plays an important role in inflammation.9,39–41 PAR-2 is widely expressed10,42 and is increased by inflammatory cytokines.39,43 In the lungs, PAR-2 is expressed in bronchial epithelial and smooth muscle cells and also in fibroblasts.10,44 PAR-2 activation increases vascular permeability,40 leukocyte recruitment,41 and secretion of proinflammatory cytokines,39,43 as well as release of MMPs from airway epithelial cells.9 By activating PAR-2, trypsin and trypsin-like enzyme tryptase can induce an increase in lung fibroblast proliferation, indicating a role in the fibroproliferative processes observed with pulmonary interstitial diseases such as BPD.10

The potential role of pulmonary trypsin in the pathogenesis of BPD raises the possibility to prevent BPD by exogenous proteinase inhibitors. Administration of exogenous {alpha}1-antitrypsin to neonatal rats can protect them from toxic effects of oxygen on the lung parenchyma and vasculature.45 Recently, in preterm infants with RDS, treatment with {alpha}1-antitrypsin to inhibit neutrophil elastase activity reduced the incidence of BPD, although the difference did not reach statistical significance.46 The investigators have later suggested that higher or more frequent doses of {alpha}1-antitrypsin may be necessary.47 On the basis of our results, the favorable effects of {alpha}1-antitrypsin may be explained, at least partially, by inhibition of pulmonary trypsin.


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In preterm infants, the development of BPD is associated with increased expression of pulmonary trypsin-2 during the early postnatal period. In the preterm lung, trypsin-2 may cause tissue damage through several mechanisms. As a powerful proteinase and MMP activator, it may initiate the tissue destruction characteristic for BPD. It is also capable of mediating inflammatory reactions, and it may play a role in the subsequent fibroproliferative response. Additional studies on treatment with exogenous proteinase inhibitors such as {alpha}1-antitrypsin for the prevention of BPD in preterm infants with RDS thus are warranted.


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  		TABLE 2. Clinical Parameters and Trypsinogen-2 Concentrations in TAF During Weeks 1 and 2

 

   ACKNOWLEDGMENTS
 
This study was supported by grants from the Wilhelm and Else Stockmann Foundation, the Foundation for the Pediatric Research, Finska Läkaresällskapet, the Helsinki University Research Funds, the Helsinki University Central Hospital Research Fund, and the Sigrid Juselius Foundation.

We thank the personnel of the neonatal intensive care unit of the Hospital for Children and Adolescents, Helsinki University Central Hospital, for their kind cooperation; Elina Laitinen for her excellent technical assistance; and Carolyn Norris, PhD, for her skillful linguistic revision of the manuscript.


   FOOTNOTES
 
Received for publication Aug 26, 2002; Accepted Feb 14, 2003.

Reprint requests to (K.C.) Hospital for Children and Adolescents, Research Laboratory, Biomedicum Helsinki, Box 700, FIN-00029 HUS, Helsinki, Finland. E-mail: katariina.cederqvist{at}helsinki.fi


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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  3. Koivunen E, Saksela O, Itkonen O, Osman S, Huhtala ML, Stenman UH. Human colon carcinoma, fibrosarcoma and leukemia cell lines produce tumor-associated trypsinogen. Int J Cancer.1991; 7 :592 –596
  4. Cocks TM, Fong B, Chow JM, et al. A protective role for protease-activated receptors in the airways. Nature.1999; 398 :156 –160[CrossRef][Medline]
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  6. Koshikawa N, Hasegawa S, Nagashima Y, et al. Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse. Am J Pathol.1998; 153 :937 –944[Abstract/Free Full Text]
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