The sialylation of bronchial mucins secreted by patients suffering from cystic fibrosis or from chronic bronchitis is related to the severity of airway infection

Monique Davril, Sophie Degroote, Pascale Humbert, Claude Galabert2, Viviane Dumur1, Jean-Jacques Lafitte1, Geneviève Lamblin and Philippe Roussel3

Unité INSERM no. 377 and 1Université de Lille 2, Place de Verdun, 59045 Lille Cedex, France and 2Hôpital Renée Sabran, Giens, 83400 Hyères, France

Received on June 16, 1998; revised on July 17, 1998; accepted on July 24, 1998

Bronchial mucins were purified from the sputum of 14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis, using two CsBr density-gradient centrifugations. The presence of DNA in each secretion was used as an index to estimate the severity of infection and allowed to subdivide the mucins into four groups corresponding to infected or noninfected patients with cystic fibrosis, and to infected or noninfected patients with chronic bronchitis. All infected patients suffering from cystic fibrosis were colonized by Pseudomonas aeruginosa. As already observed, the mucins from the patients with cystic fibrosis had a higher sulfate content than the mucins from the patients with chronic bronchitis. However, there was a striking increase in the sialic acid content of the mucins secreted by severely infected patients as compared to noninfected patients. Thirty-six bronchial mucins out of 38 contained the sialyl-Lewis x epitope which was even expressed by subjects phenotyped as Lewis negative, indicating that at least one [alpha]1,3 fucosyltransferase different from the Lewis enzyme was involved in the biosynthesis of this epitope. Finally, the sialyl-Lewis x determinant was also overexpressed in the mucins from severely infected patients. Altogether these differences in the glycosylation process of mucins from infected and noninfected patients suggest that bacterial infection influences the expression of sialyltransferases and [alpha]1,3 fucosyltransferases in the human bronchial mucosa.

Key words: fucosyltransferases/inflammation/sialic acid/sialyl-Lewis x/sialyltransferases

Introduction

Cystic fibrosis (CF), a general exocrinopathy, is the most frequent, lethal inherited disease affecting Caucasians (Welsh et al., 1995). CF is due to alterations of the CF gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (Welsh et al., 1995). CFTR is a cAMP-regulated chloride channel but has other functions such as regulation of sodium channels, outwardly rectifying chloride channels, and also transport of ATP (Boucher, 1994; Welsh et al., 1995; Devidas and Guggino, 1997). CF patients usually suffer from chronic lung infection by Pseudomonas aeruginosa leading to a progressive and destructive lung disease. The pathophysiology of the chronic lung infection is not well understood. CF respiratory cells have defective chloride secretion and elevated sodium absorption (Boucher, 1994), and lung infection may result from an alteration of the airway mucociliary clearance and/or from abnormalities in the defense mechanisms related to abnormal ion and water movements across the airway epithelium (Smith et al., 1996; Goldman et al., 1997). However, the ion concentration of airway surface liquid of patients suffering from cystic fibrosis is currently a matter of debate (Knowles et al., 1997). In addition to its role on ion movements, CFTR may influence the processing of glycoproteins and alterations of the CF gene may affect the biosynthesis of airway mucins.

Human airway mucins are high molecular weight and heavily glycosylated glycoproteins synthesized in goblet cells and bronchial glands of the human airway mucosa. They are encoded by several mucin genes and the synthesized apomucins undergo very diverse posttranslational modifications leading to hundreds of different carbohydrate chains that can be devoid of acidic residues or substituted by N-acetylneuraminic acid and/or sulfate groups (Roussel and Lamblin, 1996). These airway mucins are part of the mucociliary escalator which normally removes inhaled particles or microorganisms, and the carbohydrate chains are believed to act as recognition sites for bacteria or viruses. As a matter of fact, several neutral or sialylated carbohydrate chains analogous to carbohydrate chains of airway mucins may be recognized by different microorganisms (Roussel and Lamblin, 1996).

Altered sulfate content of airway mucins from CF patients has been reported (Roussel et al., 1975; Boat et al., 1976; Lamblin et al., 1977; Chace et al., 1985). Since the airways of these patients are usually heavily infected, especially by P.aeruginosa, the relationship of such mucin abnormalities with the primary defect of cystic fibrosis has been questioned for a long time. However, altered sulfation and glycosylation of glycoproteins secreted by CF cells in culture (Frates et al., 1983; Cheng et al., 1989; Barasch et al., 1991; Barasch and Al-Awqati, 1993) as well as hypersulfation of CF human airway mucins secreted by a xenograft model of CF airway mucosa have also been reported (Zhang et al., 1995). The lack of infection in the xenograft model suggests a link between hypersulfation of CF mucins and the primary defect of the disease. Nevertheless, there is so far no evidence for an involvement of sulfate groups in the ability of airway mucins to bind to P.aeruginosa.

More recently, much attention has been paid to the precocity of lung inflammation in CF patients (Birrer et al., 1994; Konstan et al., 1994; Bonfield et al., 1995a,b) and to the excessive inflammatory response of CF mice to bronchopulmonary infection with P.aeruginosa (Heeckeren et al., 1997). Therefore, the question of whether or not mucosal alterations due to inflammation pave the way for subsequent bacterial colonization is a matter of debate.

Inflammation as such may modify certain glycoproteins. Quantitative alterations of acute phase glycoproteins synthesized in the liver in relation to inflammation are well known but several recent reports have indicated possible glycosylation alterations of acute phase glycoproteins such as an increased expression of sialyl-Lewis x epitopes in relation to the secretion of cytokines (De Graaf et al., 1993; Havenaar et al., 1995). Many carbohydrate chains containing the sialyl-Lewis x epitope have been isolated from mucins secreted by patients suffering either from cystic fibrosis or from chronic bronchitis (Lamblin et al., 1984; Breg et al., 1987; Van Halbeek et al., 1988; Klein et al., 1993). One may therefore raise the question of an alteration of the glycosylation process in the airway mucosa of severely infected patients.

In the present work, we purified 38 mucins secreted by patients, severely infected or not, suffering either from cystic fibrosis or from chronic bronchitis and we compared their sialic acid and sulfate contents, as well as the sialyl-Lewis x expression. The mucins from infected patients were more sialylated and contained more sialyl-Lewis x epitopes than the mucins from not severely infected patients.

Results

Bronchial mucin purification and estimation of infection

Mucin isolation from the 38 samples of the patients under study (14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis) was performed by a first step of density-gradient centrifugation. Figure 1 shows typical separations obtained from the sputum of an infected patient suffering from cystic fibrosis (Figure 1A) and from the sputum of a noninfected patient suffering from chronic bronchitis (Figure 1B). In the CF sample, the high-density, orcinol-reactive fraction containing the mucins exhibits a high absorbance at 260 nm, suggesting the presence of nucleic acid (Figure 1A). The presence of nucleic acid in this mucin fraction was detected by agarose electrophoresis (Figure 2). Mucins were intensively stained by Schiff-periodate in the two samples, whereas the anodic band stained with toluidine blue and corresponding mostly to nucleic acid was only visualized in the CF sample (Figure 2).


Figure 1. CsBr density-gradient centrifugation profiles of two mucin preparations obtained from an infected patient suffering from cystic fibrosis (A and C) and from a noninfected patient suffering from chronic bronchitis (B and D). Sputum supernatants (80 mg) were submitted to a first step of density-gradient centrifugation (A and B). Fractions 1-3 were collected, treated by nucleases and glycosaminoglycan-degrading enzymes, and further submitted to a second step of density-gradient centrifugation (C and D). The purified mucins were collected in tubes 1-3. Open boxes indicate A260nm and solid circles A520nm; dotted line signifies density.


Figure 2. Comparative study on agarose gel electrophoresis of the mucin-containing fractions obtained by CsBr density-gradient centrifugation (1st step in Figure 1) of sputum supernatants from a noninfected patient suffering from chronic bronchitis and from an infected patient suffering from cystic fibrosis. The gels were stained by periodic acid-Schiff (a) and by toluidine blue (b). Mucins are visualized by periodic acid-Schiff. In contrast to the sample from the noninfected patient, the sample from the infected patient contains an anodic band stained by toluidine blue corresponding mostly to nucleic acids.

We therefore attempted to estimate an index of infection of each sample by calculating the following ratio: absorbance at 260 nm (nucleic acid estimation)/absorbance at 520 nm with orcinol (carbohydrate reaction). As shown on Figure 3, comparison of this index with the presence or absence of nucleic acid on agarose electrophoresis after the first step of density-gradient centrifugation allowed a subclassification of the 38 patients into a group of 20 infected patients (ratio above 1.1) and another group of 18 mildly (or non-) infected patients (ratio below 1.1). The first group corresponded to young CF patients (Table I, patients 1-13) and to older patients suffering from chronic bronchitis (Table I, patients 15-21). In this group of severely infected patients, all the CF patients and three CB patients (patients 15, 19, and 21) were colonized by P.aeruginosa, whereas the other patients suffering from chronic bronchitis (patients 16, 17, 18, and 20) were colonized by other bacteria (data not shown). The second group corresponded to 17 adult patients suffering from chronic bronchitis (Table I, patients 22-38) and to one CF patient (Table I, patient 14) who were not or only mildly infected (the detailed germ analyses are not shown). Patient 14, although harboring some P.aeruginosa, was mildly infected at the time his sputum was sampled.


Figure 3. Determination of the ratio A260nm (nucleic acids)/A520nm(neutral sugar) in the mucin preparations (1st step of CsBr density-gradient centrifugation) from infected (n = 20) and noninfected patients (n = 18).

After the first step of centrifugation, the mucin fractions were treated by nucleases and glycosaminoglycan-degrading enzymes to remove the contaminating nucleic acids and proteoglycans if any (Rahmoune et al., 1991). They were further purified using a second step of density-gradient centrifugation (Figure 1C,D).

Sulfate content of the 38 bronchial mucins

The sulfate content of the 38 mucins is reported in Table I. The average sulfation of bronchial mucins from patients suffering from cystic fibrosis (3.31% ± 0.25) was higher than that of mucins from the patients suffering from chronic bronchitis (2.67% ± 0.56), and the difference was significant (P = 0.024). Moreover, the average sulfation of bronchial mucins from the 20 infected patients suffering from cystic fibrosis or from chronic bronchitis (3.16% ± 0.18) was also higher than that of the mucins from the 18 noninfected patients (2.62% ± 0.15), and the difference was significant (P = 0.014).

Sialic acid content of the 38 bronchial mucins

The 38 individual mucins were also investigated for their N-acetylneuraminic acid content (Table I). Comparison of the two groups of patients according to infection showed that the mucins of the infected patients had a higher sialic acid content (mean: 7.18%) than that of the mucins from noninfected patients (mean: 3.92%; Table II). This difference is highly significant. When the two groups of patients suffering from cystic fibrosis or from chronic bronchitis were subdivided according to infection, one could observe that the sialic acid contents of the mucins from the infected patients suffering either from cystic fibrosis or from chronic bronchitis were significantly higher than those of the noninfected patients (Figure 4, Table II).

Table I. Sulfate, sialic acid and sialyl-Lewis x contents of 38 human respiratory mucins from infected or noninfected patients suffering from cystic fibrosis or from chronic bronchitis
Patient Mutations Pa, Bca A260 nm/A520nm Sulfateb Sialic acidb Sialyl-Lewis xc Phenotyped
1 CF [Delta]F508/L137R Pa 6.3 4.8 9.1 1.02 Se/Le
2 CF [Delta]F508/S1251N Pa 2.1 2.7 9.2 1.75 se/le
3 CF [Delta]F508/S1251N Pa 1.9 2.9 7.9 1.98 se/le
4 CF [Delta]F508/[Delta]F508 Pa 2.3 3.1 8.0 0.96 Se/Le
5 CF [Delta]F508/[Delta]F508 Pa 1.4 3.1 6.0 1.19 Se/Le
6 CF [Delta]F508/2184DelA Pa 1.5 5.6 5.2 1.49 Se/Le
7 CF [Delta]F508/
1717-1G->A
Pa 1.8 3.3 6.2 1.51 Se/Le
8 CF [Delta]F508/unknown Pa 4.2 3.3 8.2 0.66 se/le
9 CF N.D. Pa 1.5 3.6 4.7 0.81 Se/le
10 CF [Delta]F508/[Delta]F508 Pa + Bc 4.0 2.2 10.0 1.23 Se/Le
11 CF [Delta]F508/2143DelT Bc 2.5 2.5 8.5 1.33 Se/Le
12 CF [Delta]F508/unknown Pa + Bc 1.4 2.6 7.2 0.67 Se/Le
13 CF [Delta]F508/[Delta]F508 Pa + Bc 5.7 2.7 9.4 0.81 Se/Le
14 CF [Delta]F508-/- Pa 0.8 3.9 1.6 0.02 Se/Le
15 CB N.D. Pa 2.7 3.4 4.3 1.50 Se/le
16 CB N.D.   3.4 3.3 9.7 0.94 Se/Le
17 CB N.D.   1.7 3.2 2.0 0.82 Se/Le
18 CB N.D.   4.6 2.6 8.2 1.31 Se/Le
19 CB N.D. Pa 1.4 2.5 6.5 1.14 Se/Le
20 CB N.D.   1.8 3.3 5.8 1.03 se/Le
21 CB N.D. Pa 2.7 2.5 7.6 1.00 Se/Le
22 CB N.D.   0.6 3.2 6.8 0.94 Se/Le
23 CB N.D.   0.4 2.3 3.1 1.02 Se/Le
24 CB N.D.   0.9 4.0 1.3 0.03 Se/Le
25 CB N.D. Pa 0.6 2.4 4.7 1.44 Se/le
26 CB N.D.   0.7 2.6 3.3 0.67 se/Le
27 CB N.D.   0.4 2.1 4.0 1.26 Se/Le
28 CB N.D.   0.5 1.8 3.1 1.44 Se/Le
29 CB N.D.   0.9 2.2 4.9 0.67 Se/Le
30 CB N.D.   0.4 2.8 6.1 0.85 se/Le
31 CB N.D.   1.1 2.3 4.2 1.10 Se/Le
32 CB N.D.   0.4 2.0 3.0 1.19 Se/Le
33 CB N.D.   0.3 1.6 3.5 0.67 Se/Le
34 CB N.D.   0.4 2.5 5.8 0.90 Se/Le
35 CB N.D.   0.5 2.9 3.2 0.51 se/Le
36 CB N.D.   0.4 3.1 2.4 0.14 Se/le
37 CB N.D.   0.4 2.9 2.1 0.13 Se/Le
38 CB N.D.   0.7 2.5 7.5 1.41 Se/Le
N.D., Not determined
aPa, Presence of Pseudomonas aeruginosa; Bc, presence of Burkholderia cepacia.
bSulfate and sialic acid are expressed in % by weight.cThe reactivity of the mucin samples with the anti-sialyl-Lewis x mAb was determined in ELISA assay by measuring absorbance at 490 nm.
dThe determination of the secretor and Lewis status of each mucin is indicated in Table III.

Table II. Comparison of the sialic acid content and of the sialyl-Lewis x reactivity of the bronchial mucins from infected and noninfected patients suffering from cystic fibrosis or from chronic bronchitis
Mucin origin No. subjects Sialic acida P Sialyl-Lewis xb P
Infected (CF + CB) 20 7.18 ± 0.46 0.0001 (inf vs. non-inf) 1.16 ± 0.08 0.035 (inf. vs. non-inf)
Noninfected (CF + CB) 18 3.92 ± 0.41   0.80 ± 0.11  
Infected CF 13 7.66 ± 0.47 NS (inf CF vs. inf CB) 1.19 ± 0.12 NS (inf CF vs inf CB)
Noninfected CF 1 1.6   0.02  
Infected CB 7 6.30 ± 0.97 0.04 (inf. CB vs. non-inf CB) 1.11 ± 0.09 NS (inf. CB vs. non-inf CB)
Noninfected CB 17 4.06 ± 0.41 0.0001 (inf CF vs. non-inf CB) 0.85 ± 0.11 NS (inf CF vs. non-inf CB)
The mucins were obtained from infected (inf) or non-infected (non-inf) mucus samples from patients suffering from cystic fibrosis (CF) or from chronic bronchitis (CB).
aMean ± SEM. Sialic acid is expressed in % by weight.
bMean ± SEM. The reactivity of the mucin samples with the anti-sialyl-Lewis x mAb was determined in ELISA assay by measuring absorbance at 490 nm.

Secretor and Lewis phenotyping of the purified mucins

The secretor and Lewis phenotypes were checked before assaying the sialyl-Lewis x reactivity of the 38 mucins. Figure 5 summarizes the pathways including the fucosyltransferases Fuc-TII (secretor enzyme) and Fuc-TIII (Lewis enzyme) which are supposed to be involved in the biosynthesis of the different secretor and Lewis epitopes of human bronchial mucins.


Figure 4. N-Acetylneuraminic acid content of purified mucins from infected (inf) and noninfected (non-inf) patients suffering either from cystic fibrosis or from chronic bronchitis. Means ± SEM are indicated in Table II.

In order to establish the secretor status of the mucins, ELISA assays were used with both the anti-H mAb and the Ulex europaeus lectin which recognize the H-type 2 antigen. Anti-Lewis a and anti-sialyl-Lewis a mAbs were used to assess the Lewis status of the mucins. The simultaneous expression of H and Lewis determinants was assessed using anti-Lewis b and anti-Lewis y mAbs (see Figure 5 and Table III).


Figure 5. Biosynthesis of Lewis and H mucin epitopes. G, Galactose; Gn, N-acetylglucosamine; F, fucose; SA, N-acetylneuraminic acid; ST3, [alpha]2,3-sialyltransferase; Fuc-TII, [alpha]1,2 fucosyltransferase; Fuc-TIII, [alpha]1,3/4 fucosyltransferase.

Table III. Determination of the Secretor and Lewis phenotypes of 38 respiratory mucins
Patient H Ulex Ley Leb Lea Sia-Lea Phenotype ABO
1 CF 0.40 0.49 0.17 0.54 0.09 0.38 Se/Le O
2 CF 0.01 0.01 0.00 0.01 0.09 0.05 se/le O
3 CF 0.01 0.00 0.00 0.02 0.09 0.09 se/le O
4 CF 0.52 0.79 0.21 0.69 0.05 0.44 Se/Le O
5 CF 0.94 0.98 0.54 0.81 0.12 0.19 Se/Le O
6 CF 0.81 1.17 0.13 1.05 0.26 0.36 Se/Le O
7 CF 1.28 1.97 0.28 1.34 0.06 0.10 Se/Le O
8 CF 0.03 0.05 0.07 0.11 0.03 0.17 se/le O
9 CF 0.20 0.11 0.08 0.01 0.02 0.00 Se/le O
10 CF 0.66 0.60 0.83 0.90 0.16 0.31 Se/Le O
11 CF 0.87 0.58 0.83 1.04 0.10 0.12 Se/Le O
12 CF 0.28 0.36 0.31 0.47 0.02 0.17 Se/Le O
13 CF 0.06 0.03 0.11 0.15 0.02 0.42 Se/Le A
14 CF 0.06 0.18 0.02 0.39 0.06 0.01 Se/Le A
15 CB 1.17 1.15 0.29 0.03 0.03 0.00 Se/le O
16 CB 0.31 0.09 0.29 0.60 0.20 0.28 Se/Le O
17 CB 1.34 1.34 0.48 1.40 0.25 0.07 Se/Le O
18 CB 0.57 0.22 0.43 0.56 0.02 0.18 Se/Le O
19 CB 0.08 0.03 0.06 0.32 0.29 0.24 Se/Le A
20 CB 0.03 0.01 0.03 0.18 0.79 1.61 se/Le A
21 CB 0.02 0.00 0.07 0.26 0.14 0.44 Se/Le B
22 CB 0.27 0.17 0.20 0.41 0.02 0.13 Se/Le O
23 CB 1.21 1.34 0.28 1.05 0.22 0.24 Se/Le O
24 CB 0.22 0.19 0.05 0.65 0.20 0.03 Se/Le O
25 CB 1.82 2.07 0.18 0.01 0.02 0.00 Se/le O
26 CB 0.00 0.00 0.01 0.86 1.22 1.07 se/Le O
27 CB 1.25 1.21 0.20 0.99 0.11 0.04 Se/Le O
28 CB 1.49 1.53 0.12 1.23 0.35 0.27 Se/Le O
29 CB 0.82 0.66 0.60 0.60 0.04 0.12 Se/Le O
30 CB 0.01 0.01 0.02 0.28 0.54 1.07 se/Le O
31 CB 1.49 1.23 0.70 1.00 0.21 0.12 Se/Le O
32 CB 1.73 1.90 0.30 1.27 0.43 0.21 Se/Le O
33 CB 0.74 0.75 0.16 0.53 0.02 0.12 Se/Le O
34 CB 0.95 0.66 0.96 0.71 0.03 0.29 Se/Le O
35 CB 0.01 0.00 0.04 0.90 1.95 0.83 se/Le O
36 CB 0.03 0.11 0.20 0.00 0.00 0.00 Se/le O
37 CB 1.03 1.76 0.60 0.72 0.06 0.02 Se/Le O
38 CB 0.06 0.02 0.18 0.37 0.38 0.60 Se/Le A
The reactivity of the mucin samples with the lectin (Ulex) and mAbs was determined in ELISA assay by measuring absorbance at 490 nm.


The two patients (patients 2 and 3) whose mucins did not react with the Ulex lectin or anti-Lewis antibodies (Table III) were genotyped for the 428G-A mutation of the secretor gene (FUT2) and for the most frequent mutations of the Lewis gene (FUT3). They were characterized as homozygotes for the mutation 428G-A of the FUT2 gene and compound heterozygotes for the FUT3 gene (59T-G and 1067T-A for one allele, and 202T-C and 314C-T for the other). Therefore, the genotyping of these two patients confirmed the phenotyping of their mucins: these patients did not express active fucosyltransferase Fuc-TII or Fuc-TIII.

Sialyl-Lewis x expression in the 38 bronchial mucins

The sialyl-Lewis x reactivity of the purified mucins is presented in Table I. Comparison with the secretor and Lewis status (Table I) shows that mucins from seven patients (patients 2, 3, 8, 9, 15, 25, and 36) phenotyped as Lewis negative have a sialyl-Lewis x reactivity. This indicates that the expression of sialyl-Lewis x in these patients implies a fucosyltransferase which is different from the fucosyltransferase Fuc-TIII.

The comparison of the different groups of airway mucins is shown on Table II. The average sialyl-Lewis x reactivity of the infected samples is higher than that of noninfected samples but there is no significant difference between mucins secreted by CF patients and mucins secreted by patients suffering from chronic bronchitis.

Discussion

In the present work, we compared the sulfate and sialic acid contents as well as the sialyl-Lewis x expression of bronchial mucins from 38 patients suffering from chronic bronchitis or from cystic fibrosis.

The present data confirm the higher sulfation already reported in cystic fibrosis (Roussel et al., 1975; Boat et al., 1976; Lamblin et al., 1977; Chace et al., 1985). Several hypotheses have been postulated to explain this oversulfation which is related to the primary defect (Zhang et al., 1995). It has been suggested that, in CF cells, alkalinization of the trans-Golgi network may modify the activity of sulfotransferases (Barasch et al., 1991). Structural studies of the sulfated carbohydrate chains of bronchial mucins from a patient suffering from cystic fibrosis have shown more carbohydrate chains bearing a 6-sulfated N-acetylglucosamine than chains having a 3-sulfated terminal galactose (Lo-Guidice et al., 1994). The two sulfotransferases responsible for these sulfation reactions have been characterized and the optimal pH of the 6-sulfo-N-acetylglucosamine-transferase is higher than that of the 3-sulfo-galactosyl-transferase (Lo-Guidice et al., 1995; Degroote et al., 1997), an observation which may suggest a preferential activity of the 6-sulfo-N-acetylglucosamine-transferase in CF. However, this issue of an alkalinization of the trans-Golgi network is controversial (Seksek et al., 1996). More recently, it has been suggested that the concentration of PAPS, the sulfate donor, may be regulated in part by CFTR (Pasyk and Foskett, 1997) and therefore may influence the sulfation process. The wild-type CFTR would tend to lower the PAPS concentration in the Golgi lumen by letting PAPS leak out of the Golgi whereas the lack of CFTR in CF would increase PAPS concentration in the Golgi and therefore would favor sulfation reactions.

The sulfate content of the mucins from the infected patients was also higher than that of the mucins from the noninfected patients, and this raises the question of a possible influence of severe inflammation on the sulfation process.

Surprisingly, the sialic acid level of bronchial mucins from severely infected CF and CB patients was significantly higher than that of the mucins secreted by non-infected patients suffering from chronic bronchitis. According to our estimation of infection (Figure 3), we could observe only one noninfected CF patient (patient 14) and, as shown in Table I, the sulfate content of his bronchial mucins was high in contrast to the sialic acid content. The increased sialylation of mucins from infected patients is in agreement with our previous report showing an increased sialylation of salivary mucin glycopeptides from CF patients as compared to normals (Carnoy et al., 1993). However, in contrast to these observations, several reports in the literature indicate a decrease in the sialic acid content of secreted or membrane-bound glycoproteins from CF cells in culture (Barasch et al., 1991; Barasch and Al-Awqati, 1993; Dosanjh et al., 1994; Park et al., 1997). There are several reasons which might explain these discrepancies between glycoproteins secreted in vivo and glycoproteins obtained from cell cultures. Most immortalized respiratory cell lines studied so far may differ from typical mucin-secreting cells in the human bronchial mucosa. Some of these immortalized cell lines adopt an unusual mixed, serous and mucous, phenotype (Lo-Guidice et al., 1997). In vivo, there are differences in the sialylation pattern of the various cells of the bronchial epithelium: sialylation of the terminal galactose residues of mucins contained in goblet cells occurs mostly on the 3-OH of these residues, whereas sialylation of membrane-bound glycoproteins of the other bronchial cells occurs predominantly on the 6-OH of the terminal galactose residues (Couceiro et al., 1993). This last observation is also in agreement with all the structural works performed on carbohydrate chains of secreted human bronchial mucins, showing very little sialylation of bronchial mucins on the 6-OH of a terminal galactose (Roussel and Lamblin, 1996).

One should also consider that glycoproteins secreted by cells in culture may not have the same inflammatory environment as mucosal cells of infected patients. Even if the basic defect induces a tendency to decrease the sialylation process in cultured CF cells (Barasch et al., 1991), this tendency might be reversed in vivo by the inflammatory reaction. So far, there are indications that TNF may enhance some [alpha]2,3 sialyltransferase activity and a sialyl-Lewis x expression in colonic adenocarcinoma cell lines (Majuri et al., 1995).

In human airway mucins, sialic acid may also be attached to the 6-OH of the GalNAc residue involved in the carbohydrate-peptide linkage. However, there is a large predominance of chains with a sialic acid linked [alpha]2,3 to galactose as compared to chains with sialic acid linked [alpha]2,6 to N-acetylgalactosamine (Breg et al., 1987; Lo-Guidice et al., 1994) and, in mucins secreted by CF patients, the proportion of short sialylated chains is usually low.

Theoretically, different fucosyltransferases, Fuc-TI (H enzyme), Fuc-TII (secretor enzyme) and Fuc-TIII (Lewis enzyme), may be responsible for the fucosylation of human airway mucins, as well as the sialyl-Lewis x expression (Figure 5). The fucose content of respiratory mucins may vary according to the expression of these different fucosyltransferases, and especially according to the Lewis or H genotype of each individual. The mucins of two patients (patients 2 and 3) with blood group O (therefore expressing fucosyltransferase Fuc-TI for the biosynthesis of erythrocyte glycoconjugates) did not contain any H or Lewis epitopes (Table III). These two patients were considered as nonsecretor and Lewis negative and this was confirmed by finding inactivating mutations on the genes encoding fucosyltransferases Fuc-TII and Fuc-TIII. Moreover, the absence of H epitope on the mucins of these patients suggests that the fucosyltransferase Fuc-TI is not involved in the biosynthesis of respiratory mucins and probably implies that Fuc-TII is responsible for the fucosylation of both type 1 and 2 chains in the respiratory mucosa of secretor patients (see Figure 5).

In the present work, the sialyl-Lewis x epitope is expressed in 36 mucins out of 38 (Table I). Several carbohydrate chains bearing this epitope have already been identified in the mucins secreted by CF patients (Lamblin et al., 1984; Breg et al., 1987) and by a patient suffering from chronic bronchitis (Van Halbeek et al., 1988; Klein et al., 1993). Glycopeptides from airway mucins bearing sialyl-Lewis x epitopes bind to L-selectin (Crottet et al., 1996), and mucins in the airways may probably interact with leukocytes which are abundant in infected mucus. However, the physiological significance of the present observation, an overexpression of the sialyl-Lewis x epitope in mucins from infected mucus, is an open question.

Interestingly, this sialyl-Lewis x epitope is expressed as well in the mucins from the Lewis-positive subjects as in those from the Lewis-negative subjects (Table III). This is a strong indication that, in the human airway mucosa, a fucosyltransferase different from Fuc-TIII is also involved in the biosynthesis of the sialyl-Lewis x epitope (Figure 5). The fucosyltransferase Fuc-TIV which has been detected in human bronchial cells in culture (Emery et al., 1997) is a possible candidate to catalyze this reaction.

Links between expression of fucosyltransferases and inflammation have been suggested by several investigators. In acute phase glycoproteins synthesized by the liver, the sialyl-Lewis x epitope is expressed in response to a stimulation of the fucosyltransferase FucT-VI by cytokines (De Graaf et al., 1993; Havenaar et al., 1995; Brinkman-Van der Linden et al., 1996). This epitope is also present on cultured human endothelial cells from the umbilical vein, and its expression increases after TNF-[alpha] stimulation as well as that of Fuc-TVI (Majuri et al., 1994). This is also the case for colon adenocarcinoma cell lines where TNF-[alpha] enhances [alpha]2,3- sialyltransferase and [alpha]1,3/1,4-fucosyltransferase activities (Majuri et al., 1995).

Therefore, the overexpression of sialyl-Lewis x in mucins from severely infected patients may correspond (1) to an increased expression of an [alpha]2,3-sialyltransferase (ST3) competing with the fucosyltransferase Fuc-TII for the same substrate, the terminal Gal[beta]1,4GlcNAc chains (type 2 chains) (Figure 5), and possibly (2) to an increased expression of an [alpha]2,3-fucosyltransferase activity.

There is only a weak correlation between the sialic acid content of the mucins and the sialyl-Lewis x epitope assayed by ELISA (data not shown), and there may be several explanations for this observation. As already mentioned, N-acetylneuraminic acid in airway mucins is predominantly attached to the C3 of a terminal galactose but it may also be attached to the C6 of an N-acetylgalactosamine residue linking a carbohydrate chain to the apomucin (Roussel and Lamblin, 1996). It is also possible that the ELISA assay is not convenient when glycoproteins contain clusters of certain epitopes such as sialyl-Lewis x, or that the antibody does not detect the sialyl-Lewis x determinant with a 6-sulfated GlcNAc, a structure which is frequently expressed on certain bronchial mucins such as the mucins from CF patients (Lo-Guidice et al., 1994).

Finally, these different modifications of secreted CF mucins raise the question of their relationship with the colonization by P.aeruginosa. Respiratory and salivary mucins from CF patients have been shown to have a higher affinity for P.aeruginosa than most mucins from non-CF subjects (Carnoy et al., 1993; Devaraj et al., 1994). Several mucin-type carbohydrate epitopes, sialylated or neutral, are recognized in vitro by this microorganism (Welsh et al., 1995). There are also data concerning the involvement of sialic acid in the aggregation of P.aeruginosa by CF saliva: an increased aggregation of P.aeruginosa mediated by saliva from patients with CF has been observed and seems to be directly related to the sialic acid content (Komiyama et al., 1987).

However, the relevance of these observations to the in vivo situation is an open question. It has been shown that the abnormally high amounts of mucus secreted by CF patients might induce the expression of several genes in relation with the P.aeruginosa infection (Lory et al., 1996; Wang et al., 1996), but, at the present time, we do not know if bacteria grown in vitro have the same adhesive properties as bacteria grown in the airways of patients. One should also mention that Pseudomonas exoproducts induce mucin overproduction by acting on [kappa]B regulatory sites of mucin genes (Li et al., 1998a,b).

In conclusion, bronchial mucins from CF patients severely infected by P.aeruginosa undergo at least two biosynthetic abnormalities, (1) oversulfation which is mostly linked to the primary defect (Zhang et al., 1995), and (2) hypersialylation and overexpression of sialyl-Lewis x which are not specific for CF and which may reflect a strong inflammatory reaction of the respiratory mucosa.

In the future, it will be necessary to identify the different sialyl- and fucosyltransferases expressed in the human bronchial mucosa and to investigate the influence of cytokines on their expression. In the case of cystic fibrosis, it will be important to find out if the overexpression of certain sialylated carbohydrate determinants may favor the airway colonization by P.aeruginosa, the major problem faced by the patients suffering from this disease.

Materials and methods

Patients

Twenty-four patients suffering from chronic bronchitis and 14 patients suffering from cystic fibrosis were studied. All CF patients had a positive sweat test. The genotypes of the CF patients were: [Delta]F508/[Delta]F508 (n = 4); [Delta]F508/S1251N (n = 2); [Delta]F508/L137R (n = 1); [Delta]F508/2184DelA (n = 1); [Delta]F508/2143DelT (n = 1); [Delta]F508/1717-1G->A (n = 1); [Delta]F508/unknown (n = 2); unknown (n = 2). The ABO blood group of these patients was determined. Sputa were collected and the individual samples were kept frozen until used.

Identification of Pseudomonas aeruginosa and Burkholderia cepacia in the sputa

The sputum samples were plated on the following media: Drigalski (for growth of Pseudomonas aeruginosa) and selective differential OFPBL agar (for growth of Burkholderia cepacia) containing 9.4 g oxidation-fermentation basal medium, 15.0 g Bacto agar, 10.0 g lactulose (all from Difco Lab.), 300,000 U polymyxin B sulfate and 200 U bacitracin (both from Sigma) per liter. Isolates were identified by standard biochemical procedures (API 20 NE, Bio Mérieux, Marcy-l'Etoile, France).

Preparation of bronchial mucins

The collected sputa were thawed, diluted 1:12 with deionized water containing 0.02% sodium azide, stirred overnight at 4°C and centrifuged at 3000 × g for 30 min. The supernatants were dialyzed and freeze-dried (Slayter et al.,1984). Aliquots of the above materials were dissolved by stirring at 4°C for 48 h in 16.7 mM sodium phosphate buffer, pH 7.2, containing 33 mM NaCl, 0.02% sodium azide, and 42% CsBr. Insoluble materials, if any, were removed by centrifugation as above and the supernatants were submitted to a first step of density-gradient centrifugation at 43,000 r.p.m. for 72 h at 10°C, as described previously (Houdret et al., 1986). Fractions (1 ml) were collected from the bottom of each centrifuge tube and aliquots were diluted 1: 4 with deionized water.

The diluted aliquots were measured for absorbance of nucleic acid at 260 nm and for hexose content by an automated orcinol assay (Demaille et al., 1965). Densities were also measured by weighing aliquots of each fraction. The high-density (d [ge] 1.46 g/ml) fractions were pooled and exhaustively dialyzed against deionized water and freeze-dried. The crude mucin preparations were subjected to a purification procedure which first involved nucleic acid removal. Aliquots were dissolved in 62 mM sodium tetraborate buffer, pH 8.8, containing 1.25 mM CaCl2 and 0.05% BSA, in the presence of 50 µM Pefabloc SCr (Pentapharm, Basel, Switzerland). DNase (nuclease, micrococcal, Sigma Chemical Co., St. Louis, MO) and RNase (type I-AS from bovine pancreas, Sigma) were added in amounts of 100 mU and 420 mU, respectively, per mg of crude mucin, and the mixtures were gently stirred for 24 h at 37°C. The same amounts of both enzymes were added again and the reaction was proceeded for another 24 h. The solutions were dialyzed and lyophilized. The nuclease-treated materials were then dissolved in 200 mM Tris-acetate buffer, pH 7.5, containing 2 mM CaCl2, and the following enzymes were added: hyaluronate lyase from S.hyalurolyticus (type IX, Sigma), chondroitinase ABC from P.vulgaris (Seikagaku, Tokyo, Japan), heparinase III from F.heparinum (Sigma) in amounts of 100 mU, 50 mU, and 50 mU, respectively, per mg of nuclease-treated material. The mixtures were stirred overnight at 37°C and freeze-dried.

A second step of CsBr density-gradient centrifugation was then performed in the same conditions and the collected fractions were assayed as above. The high density-, hexose-rich materials which contained the purified mucins were recovered, dialyzed, and freeze-dried.

Agarose gel electrophoresis

Aliquots of the purified mucins (400 µg) were subjected to agarose gel electrophoresis in veronal buffer, pH 8.2, as described previously (Rahmoune et al., 1991). Slides were stained for protein with amidoblack, for carbohydrate with periodic acid-Schiff reagent and for acidic components with toluidine blue.

Analytical procedures

Total sialic acid content was assessed, after hydrolysis with 0.1 M H2SO4 for 30 min at 80°C, by the thiobarbituric acid method (Aminoff, 1961) and expressed as N-acetylneuraminic acid. Sulfate determination was performed by high-pressure anion-exchange chromatography after hydrolysis with 1 M HCl for 5 h at 100°C (Lo-Guidice et al., 1994).

Enzyme-linked immunosorbent assay

The purified mucin samples were dissolved in PBS, pH 7.2, and aliquots (25 ng in 100 µl PBS) were coated on 96-well Maxisorp immunoplates (Nunc, Roskilde, Denmark) overnight at 4°C. Unbound sites were blocked with BSA (1% in PBS) for 2 h at room temperature (RT). After washing with PBS, the following mouse mAbs, anti-H type 2 (Dako Corp., Carpinteria, CA) diluted 1:100; anti-Lewis b (Emery et al., 1995) diluted 1:1000; anti-Lewis a (Seikagaku) diluted 1:16,000; anti-Lewis y (Seikagaku) diluted 1:100; anti-sialyl-Lewis a (Seikagaku) diluted 1: 2000; anti-sialyl-Lewis x (Kamiya Biomed. Comp., Seattle, WA) diluted 1:400, all in PBS containing 1% BSA, were added and left for 1 h at RT. The wells were washed once with PBS-0.1% Tween-20 and three times with PBS. The peroxidase-conjugated goat antibody against mouse IgG (H+L) (Jackson Immuno Research Lab., Westgrove, PA) diluted 1:2000 in PBS-1% BSA was added and left for 90 min at RT. After successive washings as above, the plates were developed with o-phenylenediamine (Sigma) at 1 mg/ml in 0.1 M sodium phosphate-citrate buffer, pH 5.5, in the presence of 0.03% H2O2. The reaction was stopped with 1 M HCl, and the plates were read at 490 nm on a Bio-Rad model 3550 microplate reader (Bio-Rad Lab., Hercules, CA). Ulex europaeus lectin biotin-labeled (Seikagaku) and peroxidase-conjugated streptavidin (Pierce, Rockford, IL) were diluted 1:2000.

Genotyping of fucosyltransferases FUT2 and FUT3

Methods based upon polymerase chain reaction and restriction fragment length polymorphism were used to detect mutations of the FUT2 gene (coding for the Secretor enzyme) and of the FUT3 gene (coding for the Lewis enzyme) (Kelly et al., 1995; Ørntoft et al., 1996).

Statistical analysis

Results are presented as means ± SEM. Data were compared using Mann-Whitney U. A P < 0.05 was considered significant.

Acknowledgments

We thank M-P.Ducourouble, M-C.Houvenaghel, M.Luyckx, and C.Vandeperre for excellent technical assistance. This investigation was supported by the Association Française de Lutte contre la Mucoviscidose.

Abbreviations

CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CB, chronic bronchitis; Fuc-T, fucosyltransferase; FUT, fucosyltransferase gene; Se, secretor; se, non secretor; Le, Lewis positive; le, Lewis negative; Lea, Lewis a; Sia-Lea, sialyl-Lewis a; Leb, Lewis b; Lex, Lewis x; Ley, Lewis y; ST, sialyltransferase; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; BSA, bovine serum albumin; PAPS, 3[prime]-phosphoadenosine 5[prime]-phosphosulfate; TNF, tumor necrosis factor; DNase, deoxyribonuclease; RNase, ribonuclease; PBS, 0.02 M phosphate-buffered saline; RT, room temperature.

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