INTRODUCTION

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Mucins, the major components of the mucus covering of the airway epithelia, are complex glycoproteins with high molecular weights and heavy glycosylation (1). The human respiratory mucins belong to a broad family of different mucin peptides characterized by diverse carbohydrate side chains. With the development of monoclonal antibodies (mAbs), several studies have been done to assess the relationship of lung diseases and mucins (2). Previous studies suggest that the mucin antigen in the serum of patients with pulmonary diseases is largely of pulmonary origin (2). Serum mucin levels are increased in cystic fibrosis (CF) patients, and serum levels of mucin-associated antigen correlate with the pulmonary inflammation in these patients (3, 4). Kohno and colleagues reported that KL-6 antigen, a human MUC-1 mucin expressed on Type II pneumocytes, was a sensitive serum marker for evaluating the disease activity of interstitial pneumonia and pulmonary fibrosis and the extent of pulmonary tuberculosis (5, 6). However, no previous study has been published of serum levels of mucin-associated antigen in patients with acute respiratory distress syndrome (ARDS).

ARDS represents a severe, diffuse pulmonary inflammation and injury caused by a wide variety of insults (7, 8). Although the sequence of events leading to ARDS is complex, a final common pathway involving a variety of inflammatory cells and mediators ultimately results in disruption of the alveolocapillary membrane (7, 8). The permeability of pulmonary endothelium and epithelium increases with loss of the macromolecular barrier, leading to accumulation of neutrophils and protein-rich fluid in the alveolar spaces (9). The leakage of air space surfactant protein-A (SP-A) into the serum has been demonstrated in patients with ARDS (10). With evidence of severe barrier damage and increased serum mucin levels in reported pulmonary inflammatory diseases, we investigated whether serum mucin levels are also increased in patients with ARDS. The serum mucin antigen levels were quantified with a double-sandwich ELISA method (11, 12), using a specific mAb, 17Q2. We sought correlations between serum mucin levels and such clinical parameters as static respiratory-system compliance, blood oxygenation, and lung injury scores (LIS) in ARDS patients. In addition, we investigated the distribution of serum mucin molecular sizes with gel-filtration electrophoresis.

	METHODS

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Subjects and SampIe Collection

Five groups of patients were included in the study. After informed consent was obtained, serum samples (10 ml) were collected and stored in a freezer at 70° C until analysis. Group 1 consisted of 59 nonsmoking control subjects (age: 47.6 ± 2.0 yr [mean ± SEM]) who had no pulmonary symptoms or cardiopulmonary diseases. Group 2 consisted of 29 chronic smokers (age: 42.0 ± 2.8 yr) who had had no acute respiratory symptoms for 1 mo prior to blood collection. The mean smoking history of this group was 16 pack-yr. Group 3 consisted of 28 patients who were admitted for acute exacerbation of chronic bronchitis and other pulmonary diseases. All 28 patients in Group 3 had fever and leukocytosis. Nine patients in this group (age: 70.7 ± 2.1 yr) had acute exacerbations of chronic bronchitis due to infection; six patients (age: 69.2 ± 5.2 yr) had community-acquired pneumonia; six patients (age 56.2 ± 7.2 yr) had bronchiectasis with infection; five patients (age: 44 ± 11 yr) had pulmonary tuberculosis; and two patients (aged 83 and 62 yr, respectively) had idiopathic pulmonary fibrosis (IPF) with secondary infection. Group 4 consisted of five patients (age: 70.4 ± 7 yr) admitted for acute cardiogenic lung edema. All five had typical findings on their chest radiographs, distended jugular veins, diffuse crackles without a history suggesting pulmonary infection, increased pulmonary capillary wedge pressures ( 15 mm Hg), and low left ventricular ejection fractions (< 30%) upon echocardiographic examination. Group 5 consisted of 13 patients (age: 58.6 ± 5.2 yr) with hypoxemia of acute onset, bilateral pulmonary infiltrates on chest radiographs, no clinical evidence of left atrial hypertension, and diagnoses of ARDS made according to recommended criteria (7). These 13 patients were enrolled in the study when they developed ARDS in the intensive care unit (ICU) after admission, or when diagnosed with ARDS at the time of admission. In Group 5, blood was sampled on the day on which patients were enrolled in the study and then every other day. Blood-gas, static respiratory-system compliance, and ventilator settings were recorded each time blood was sampled. A chest radiograph was taken within 1 h before or after blood sampling. Static respiratory-system compliance was determined as the quotient of the measured expired tidal volume (VT) with the gradient of the plateau airway pressure to end-expiratory airway pressure during mechanical ventilation in the control mode with a Puritan-Bennett 7200ae ventilator (Puritan-Bennett Corp., Carlsbad, CA) with the patient under sedation. Intrinsic positive end-expiratory pressure (PEEPi), as measured with the end-expiratory airway occlusion method (13), was taken into account in calculating static respiratory system compliance. All ARDS patients had an LIS  2.5 (14) at admission. The clinical variables of the ARDS patients are summarized in Table 1.

Quantitation of Serum Mucin-associated Antigen

The concentration of mucin-associated antigen in serum was quantified with a double-sandwich ELISA method (11, 12). Monoclonal antibody 17Q2 recognizes the granules in human airway mucous cells and high-molecular-weight mucous glycoproteins purified from human airway secretions (11, 15). The specificity of this mAb has been assessed with immunohistochemistry and Western blot analysis (11). The "standard" mucin antigen used was purified from pooled sputa of patients with different blood types, and the purity of the standard mucin was analyzed as previously described (12, 16). All serum samples were diluted 20-fold with a phosphate-buffered saline (PBS)-Tween 20 (0.05%) buffer. Aliquots of normal saline as controls, and aliquots of standard mucin (0 to 4 ng of protein per well), were included with each microtiter plate. If the mucin concentration of a sample was > 4 ng/well, the sample was diluted and analyzed again. The results were expressed as nanograms of mucin protein per milliliter of serum sample. All samples were assayed in duplicate, and the mean value was used in the statistical analysis of the study data. The interassay coefficient of variation (CV) was 6.8%, and the intraassay CV was 5.0% (12).

Gel-filtration Chromatography

Endogenous protease activity in the bronchoalveolar fluid may cleave mucins to smaller molecules (3). To understand the molecular-size distribution of the mucin-associated antigens detected in serum, sera from three patients with ARDS, three patients with stable chronic bronchitis, and three normal subjects were subjected to gel-filtration chromatography with a method described previously (3). Briefly, 1 ml of serum was treated with an equal volume of 2× sodium dodecyl sulfate (SDS) sample buffer containing 5% -mercaptoethanol in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 20 mg/ml aprotinin), and subsequently chromatographed on a Sepharose CL-4B column (1 × 50 cm) (Pharmacia, Uppsala, Sweden). The elution buffer was a PBS-based solution containing 0.1% SDS and 0.5% -mercaptoethanol. The ELISA method was used to quantify mucin antigen in each fraction.

Statistical Analysis

In patients with ARDS, the peak levels of serum mucin were used for statistical analysis. For comparison of the mean value of mucin concentration or percentage of the void volume fraction among different study groups, we performed an analysis of variance (ANOVA), followed by use of the Student-Newman-Keuls test to compare one group with another. Because serum mucin concentrations, static respiratory compliance, log(Pa_O2/FI_O2), and LIS values were serially measured in patients with ARDS, they could be analyzed as longitudinal data and modeled with a random-effect model (17). In order to elucidate whether there exists a significant time trend in the relationship between serum levels of mucin-associated antigen and the severity of pulmonary injury (indicated by static respiratory compliance, log (Pa_O2/FI_O2), or LIS), a generalized linear mixed model (17), using the SAS Mixed Procedure (SAS Institute, Inc., Cary, NC) (18), was used to assess the relationship. Estimation of the coefficient of regression was based on maximum likelihood (17). The level for statistical significance was set at p < 0.05.

	RESULTS

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The serum level of mucin-associated antigen measured in normal subjects was 9.9 ± 0.8 ng/ml (mean ± SEM, n = 59), that in chronic smokers was 12.7 ± 1.6 ng/ml (n = 29), that in patients with chronic bronchitis and other pulmonary diseases was 21.8 ± 1.9 ng/ml (n = 28), that in patients with acute cardiogenic lung edema was 9.0 ± 3.1 ng/ml (n = 5), and that in patients with ARDS was 53.8 ± 6.6 ng/ml (n = 13). The serum mucin levels for the individuals included in each of these five experimental groups are shown in Figure 1. Sera of the ARDS group contained a statistically significantly higher concentration of mucin-associated antigen than did sera of the four other groups (p < 0.05). Furthermore, patients with chronic bronchitis and other pulmonary diseases had increased serum levels of mucin-associated antigen as compared with normal subjects and chronic smokers (p < 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Distribution of serum mucin-associated antigen levels in five groups of patients. Each patient is represented by a single dot. A horizontal line indicates the mean value in each group.

The first measured mucin levels in survivors and in nonsurvivors of ARDS were 47 ± 5.7 ng/ml and 45 ± 6.9 ng/ml, respectively. The mucin level of the four survivors prior to their discharge from the ICU was 27.5 ± 6.2 ng/ml, and the mucin level of the nine nonsurvivors prior to their death was 48 ± 6.2 ng/ml. Although mucin levels in nonsurvivors were higher than those in survivors, no attempt was made to apply a test of significance for these observations because the sample size was relatively small.

To further understand the relationship between serum levels of mucin-associated antigen and the severity of pulmonary injury, we performed serial examinations of serum mucin antigen in patients with ARDS. Statistical analysis, using the generalized linear mixed model, showed an inverse correlation between serially measured serum mucin levels and static re-spiratory-system compliance (p = 0.021). A significant inverse relationship was also found between sequential serum mucin levels and log(Pa_O2/FI_O2) (p = 0.016). There was a significant time trend for a positive association between sequential serum mucin levels and LIS (p = 0.019).

As shown in Figure 2, the gel-filtration pattern of mucin-associated antigen in ARDS sera was more polydispersed. The distributions ranged broadly, from fraction 20 to fraction 60. The gel-filtration pattern of serum mucin-associated antigen in patients with stable chronic bronchitis was relatively confined, occurring from fraction 20 to fraction 40. The gel-filtration pattern of serum mucin-associated antigen in normal subjects was essentially similar to the pattern seen in patients with stable chronic bronchitis. We analyzed the high-molecular-weight mucin fraction of each group by measuring the amount of mucin in the void volume and dividing the result by the total amount of mucin. The fraction of high-molecular-weight mucin in patients with ARDS was 15.6 ± 1.7% (mean ± SEM) (n = 3), which was statistically significantly smaller than those of patients with stable chronic bronchitis (39.6 ± 2.5% [n = 3]) and normal subjects (42.0 ± 3.0% [n = 3]) (p < 0.05) (Table 2). These results indicate the existence of a relatively large amount of low-molecular-weight mucin-associated antigens in patients with ARDS as compared with normal subjects and patients with stable chronic bronchitis.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Fractionation of serum from a patient with ARDS (open circles) and another patient with stable chronic bronchitis (closed rhombus) by gel filtration on Sepharose CL-4B. Aliquots of each fraction were assayed with an ELISA for mucin-associated antigen. The concentration of mucin-associated antigen in the serum of the ARDS patient was much higher, and the distribution was more polydispersed, than in the patient with stable chronic bronchitis. This result implies that smaller sized mucin-associated antigens were present and relatively abundant in patients with ARDS. This may in part have been due to the degradation of mucins by endogenous proteolytic activity in the patient with ARDS. (The ordinate indicates the concentration of mucin in each fraction. Arrowhead indicates the void-volume peak.)

	DISCUSSION

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Mucins are major components of airway mucus secreted by bronchial epithelial mucous cells (goblet cells and submucosal glands). Their major function is assumed to be lubrication and protection of the epithelium (1). Under normal conditions, the alveolar-airway barrier consists of tight intercellular junctions that allow passage only of water and electrolytes. Native mucin is probably too large and possesses too many negative charges to be absorbed through normal respiratory tight junctions. In patients with ARDS, histopathologic studies show variable epithelial damage, ranging from cytoplasmic swelling to total cellular destruction and hyaline-membrane-covered basement membrane (8). Bronchoalveolar lavage fluid (BALF) analysis shows an increase in inflammatory cells (especially neutrophils) and serum protein constituents in ARDS patients (9). Doyle and colleagues have shown that SP-A leaks into the serum of patients with ARDS (10). These findings indicate that in ARDS, the alveolar epithelium and/or the distal bronchiolar epithelium have an increased permeability to protein (i.e., a leaky air-blood barrier), and that bidirectional protein flux occurs. In the present study, we found increased serum levels of mucin-associated antigen in patients with ARDS.

The increased serum mucin levels in ARDS patients could be due to the increased permeability of the alveolar or distal bronchiolar epithelium; however, serum concentrations of mucin-associated antigen may depend on a number of other interrelated factors, including the rates of mucin production and degradation within the airway or serum. Neutrophils accumulate in pulmonary interstitial and alveolar fluid, and have been thought to play a major role in the mechanism of lung injury (7). Release of protease, oxygen radicals, and other secretagogues by neutrophils and other inflammatory cells may increase mucus production by goblet cells and submucosal glands (19). Previous studies have also demonstrated proteolytic fragmentation of mucins in the respiratory tract secretions of patients with CF (3, 20). Degradation of high-molecular-weight breast-tumor sialoglycoproteins by serum proteases, resulting in low-molecular-weight components, has also been described (21). These findings are consistent with our finding that serum mucin-associated antigens in patients with ARDS are more dispersed, of lower molecular weight, and greatly increased in their total amount (Figure 2). The protease activity in ARDS bronchoalveolar fluid may cleave mucin to smaller molecules, and thus permit mucin-associated antigen to pass more easily through the damaged air-blood barrier of patients with the disease, from the alveolar and bronchial air spaces into the bloodstream. These results suggest that increased serum mucin levels in patients with ARDS result from increased production and protease cleavage of mucins, and from increased airway permeability.

Our study had some limitations. First, the study groups did not include patients without parenchymal lung disease who were undergoing mechanical ventilation, or patients with parenchymal lung disease severe enough to require mechanical ventilatory support. We do not know whether mucin levels in ARDS are higher than those in severe pneumonia. Second, Robinson and colleagues (3) reported that the mean serum mucin level in 25 patients with CF, as measured with the ELISA method and mAb 17B1, was 13,853 ng/ml, which was much higher than that of our patients with ARDS. The reason for this extremely high serum mucin level is unknown. Previously reported serum mucin levels in different disease groups have also been varied (4). However, with the use of different assay systems, different mAbs, and different ethnic groups, it is impossible to compare these different results. Third, serial measurements in our study were performed only in the ARDS group. Whether serial measurements of serum mucin levels are associated with lung damage in cardiogenic lung edema needs further study. In the present study, we found that patients with pulmonary infections had higher serum levels of mucin-associated antigen than did smokers and normal subjects. We do not know whether the serum mucin levels declined after the infection was controlled.

In conclusion, in our experience, serum levels of mucin-associated antigens were increased in patients with ARDS. The increased levels of mucin in these patients' serum may be attributed to increased mucin production and degradation, and to increased airway permeability. Sequential measurements of serum mucin levels in patients with ARDS was inversely correlated with static respiratory-system compliance and log(Pa_O2/ FI_O2). The time-sequence of serum mucin levels was correlated with the time-sequence of the LIS. The overall biologic and clinical significance of increased serum mucin antigen levels in patients with ARDS is not understood. However, serial measurements of mucin levels in patients with ARDS may be worth further investigation.