American Journal of Respiratory and Critical Care Medicine

To examine the differential properties of mucous glycoproteins, we produced hypertrophic and metaplastic changes in goblet cells of rat nasal epithelium by intranasal instillation of ovalbumin (OVA) in OVA-sensitized rats, and by intranasal lipopolysaccharide (LPS) instillation. The epithelial mucosubstance was quantitatively examined by alcian blue–periodic acid–Schiff (AB–PAS) and lectin histochemistry. The newly produced mucin after OVA challenge or LPS instillation contained a high amount of sulfomucin and a low amount of neutral glycoprotein: LPS-induced mucin contained more sulfomucin (70.1% of total) and less neutral glycoprotein (8.6%) than OVA-induced mucin (sulfomucin, 33.6%; neutral glycoprotein, 41.8%; p < 0.01). Four of the lectins stained some of the mucosubstance, indicating the presence of galactose-N-acetylgalactosamine, α 2,3- and α 2,6-linked sialic acid–galactose, and fucose residues. After LPS instillation, the reactivity was higher for galactose-N-acetylgalactosamine (64.8% of total) and α 2,3-linked sialic acid–galactose (75.8%) than after saline instillation (3.5 and 19.1%, respectively) or OVA challenge (5.8 and 32.3%; p < 0.05). OVA challenge did not induce the alteration of terminal sugar residues. A 2-fold increase in mucin mRNA (rat Muc5ac) expression was induced after LPS instillation or OVA challenge, compared with animals treated with saline instillation (p < 0.05). These results indicate that mucin mRNA expression (for peptide backbone) increases similarly after LPS instillation or OVA challenge; however, carbohydrate compositions of newly produced mucin are different between the two groups.

Keywords: allergic inflammation; goblet cell; lectin; lipopolysaccharide; mucus

Airway mucus plays an important role in host defense mechanisms as a physicochemical barrier to bacteria, viruses, inhaled particles, and gases. The major components of mucus are mucous glycoproteins (mucins), which are secreted by epithelial goblet cells and submucosal glands. Mucins are large heterogeneous macromolecules, containing oligosaccharide chains attached to peptide backbones encoded by several MUC genes. Mucin carbohydrate chains are highly variable and contribute to the great heterogeneity of mucus. Twelve human mucin genes have been identified throughout the respiratory, gastrointestinal, and reproductive tracts, and MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, and MUC8 are expressed in airway epithelium (1-4).

Hypersecretion of mucus is a common characteristic of airway inflammation, such as rhinitis, sinusitis, tracheobronchitis, asthma, and cystic fibrosis. Mucin differs in the sugar groups and the extent of sialation and sulfation of the oligosaccharide structure in different hypersecretory conditions (5, 6). The alteration of these oligosaccharide structures or peptide backbones may affect the physicochemical properties of mucus in disease. It may have biological functions including host defense by entrapping inhaled bacteria or viruses and preventing their attachment to epithelium. However, little is known about the disease-related changes of mucin character and mucin gene expression in airway inflammation, and the biological roles of these changes are still unclear.

In a previous study, we produced allergic inflammation and lipopolysaccharide (LPS)-induced inflammation in rat nasal epithelium. Hypertrophic and metaplastic changes of goblet cells were induced by intranasal instillation of ovalbumin (OVA) in OVA-sensitized animals (7), and by intranasal LPS instillation (8). A similar increase in intraepithelial mucosubstance occurred 24 h after 3 d of OVA or LPS instillation. We hypothesized that the extent of sialylation and sulfation of the terminal sugar residues, and mucin gene expression, are different between mucins produced by allergic inflammation and by LPS stimulation.

In the present study, we evaluated (1) the amount of sialomucin and sulfomucin by alcian blue–periodic acid–Schiff (AB–PAS) histochemistry, (2) the terminal sugar residues by lectin histochemistry, and (3) mRNA expression of the rat Muc5ac gene in rat nasal epithelium, produced by allergic inflammation or LPS stimulation. The characterization of the properties of mucin will be useful in understanding the alterations in association with airway diseases, and in elucidating the mechanism through which mucus protects the airways.

Intranasal Instillation with Lipopolysaccharide

Male Fischer 344 rats (specific pathogen free; Japan SLC, Shizuoka, Japan) were used at 9 to 10 wk of age in this study. The rats were anesthetized with ether and 0.1 ml of saline containing 0.1 mg of LPS from Escherichia coli 0111:B4 (Sigma, St. Louis, MO) was instilled into both airways of the nasal cavity once a day for 3 d. The instillation methods and tissue preparations were the same as previously reported (7). The instillate was deposited as a bead of fluid on the external nares and the rats were allowed to aspirate it. Some rats were instilled with saline as controls.

Sensitization and Intranasal Challenge with Ovalbumin

Sensitization and challenge of rats were performed as previously reported (7). Briefly, rats (6–7 wk old) were immunized by an intraperitoneal injection of 0.1 ml of saline containing 200 μg of OVA (Grade V; Sigma) and 10 mg of Al(OH)3 on Days 1, 2, 3, and 11. Heat-killed Bordetella pertussis bacilli (1010 in 50 μl of saline; Wako Pure Chemical Industries, Osaka, Japan) were given by foot pad injection on Day 1 as an adjuvant.

On Day 19, rats were anesthetized with ether and 0.1 ml of saline containing 10 mg of OVA was instilled into both airways of nasal cavity once a day for 3 d. The instillation methods were the same as described above.

Tissue Preparations

Twenty-four hours after the last intranasal instillation with LPS or OVA, rats were killed with an intraperitoneal overdose of sodium pentobarbital. The head of each rat was removed and fixed in 10% neutral buffered formalin for 3 d, and then decalcified in 5% trichloroacetic acid for 7 d. The nasal cavity was transversely sectioned at the level of the incisive papilla of the hard palate (7). The tissue block was embedded in paraffin.

Morphometry

Paraffin sections (5 μm thick) were stained with alcian blue–periodic acid–Schiff reagent and hematoxylin (AB–PAS–H) or subjected to lectin histochemistry. The percent area of AB–PAS- or lectin-stained mucosubstance in the epithelium was determined with an image analyzer (SP 500; Olympus, Tokyo, Japan). The area of the nasal epithelium was outlined and the image analyzer determined the area of the AB–PAS- or lectin-stained mucosubstance within this reference area. The percent area of mucosubstance in the nasal epithelium was calculated over 2 mm (1 mm for each side of the nasal septum times 2) of the basal lamina at the center of the septal cartilage.

AB-PAS Histochemistry

To distinguish the mucosubstance produced by goblet cells, paraffin sections (5 μm thick) were stained with AB–PAS–H according to three different methods after Jones and Reid (9): (1) AB–PAS–H staining with the AB at pH 2.6, (2) AB–PAS–H staining with the AB at pH 2.6 under sialidase digestion, and (3) AB–PAS–H staining with the AB at pH 1.0. For sialidase treatment, sections were incubated with a 1:4 dilution of sialidase (Vibrio cholerae neuraminidase, 1 U/ml; Boehringer Mannheim Yamanouchi, Tokyo, Japan) in 0.4% CaCl2 at 37° C for 12 h before AB–PAS–H staining. Four types of mucosubstance were characterized by these staining methods: neutral glycoprotein and acid glycoproteins (sialidase-sensitive sialomucin, sialidase-resistant sialomucin, and sulfomucin). Neutral glycoprotein stains with PAS and acid glycoproteins stain with AB by AB (pH 2.6)– PAS staining. By AB (pH 2.6)–PAS with sialidase digestion, neutral glycoprotein and sialidase-sensitive sialomucin stain with PAS, whereas sialidase-resistant sialomucin and sulfomucin stain with AB. By AB (pH 1.0)–PAS, neutral glycoprotein and sialomucin stain with PAS, and sulfomucin stains with AB. The percent area of each mucosubstance in total was determined with the image analyzer.

Lectin Histochemistry

The lectins used in this study and their carbohydrate-binding specificities are listed in Table 1. Paraffin sections (5 μm thick) were incubated in 0.3% H2O2 in methanol for 20 min to inhibit endogenous peroxidases. After washing in 0.01 M phosphate-buffered saline (PBS), nonspecific lectin binding was blocked by a 10-min incubation in 1% bovine serum albumin. The sections were then incubated with biotinylated lectin (20 μg/ml; EY Laboratories, San Mateo, CA) in PBS for 30 min at room temperature. The lectin-treated sections were washed with PBS and were incubated with avidin–biotin–peroxidase complex (ABC) reagent (Vector Laboratories, Burlingame, CA) for 30 min. Peroxidase activity was visualized by a 5-min incubation in diaminobenzidine (DAB)–H2O2 solution (20 mg of DAB and 100 μl of 3% H2O2 in 100 ml of 0.05 M Tris-buffered saline, pH 7.6). The sections were counterstained with 1% methyl green. Controls included omission of biotinylated lectin, omission of the ABC reagent, and the use of lectins preincubated with a 0.2 M concentration of the specific inhibitory sugar (Table 1) for 30 min before staining.

Table 1.   LECTINS USED FOR CHARACTERIZATION OF RAT NASAL MUCUS

Abbreviation Lectin Carbohydrate Specificity Inhibitory Sugar
PNA Arachis hypogaea Galβ1,3GalNAc > α- and β-Gal Lactose
MAL-II Maackia amurensis II αNeuNAc2,3Gal Sialic acid
SNA Sambucus nigra αNeuNAc2,6Gal Sialic acid
UEA-I Ulex europaeus I α-l-Fuc α-l-Fucose
DBA Dolichos biflorus GalNAcα1,3GalNAc>>αGalNAc N-Acetylgalactosamine
PSA Pisum sativum αMan > αGlc = GlcNAc α-Methyl-mannose

Definition of abbreviations: Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; Glc = glucose; GlcNAc = N-acetylglucosamine; Man = mannose; NeuNAc = N-acetylneuraminic acid.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction

Rat nasal septal epithelium was homogenized in a denaturing solution containing 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. The RNA was prepared according to the single-step method of Chomczynski and Sacchi (10), and treated with DNase. One microgram of total RNA was reverse transcribed, and then the cDNA was amplified by polymerase chain reaction (PCR), using the GeneAmp RNA PCR kit (Takara Biochemicals, Tokyo, Japan) according to the manufacturer instructions. The rat Muc5ac cDNA was amplified with the sense primer 5′-GGCCAATGCGGCACTTGTACCAA-3′ and the antisense primer 5′-TCT GGACAGAAGCAGCCCTCTGA-3′. The rat β-actin cDNA was amplified with the sense primer 5′-AGAAGAGCTATGAGCTGC CTGACG-3′ and the antisense primer 5′-CTTCTGCATCCTGT CAGCCTACG-3′. The PCR products were resolved in a 2% agarose gel containing ethidium bromide (0.5 μg/ml). Southern blotting of the PCR products was then carried out with internal oligonucleotide probes specific for the Muc5ac and β-actin cDNAs.

Statistics

All data are expressed as means ± SD. The difference between variables was analyzed by the Mann–Whitney test. Probability values of p < 0.05 were considered significant.

Intraepithelial Production of Mucus

Intranasal instillation of LPS for three consecutive days induced hypertrophic and metaplastic changes of goblet cells in nasal septal epithelium 24 h after the last instillation. When OVA-sensitized rats were challenged with OVA on each of three consecutive days, similar changes of goblet cells occurred 24 h after the last challenge. Only a few goblet cells were observed in control groups (untreated control, saline-instilled, and sham-sensitized rats challenged with saline or OVA, and OVA-sensitized rats challenged with saline). Quantitative changes in the percent area of mucosubstance in this epithelium are described in Figure 1. Intraepithelial mucosubstance increased significantly after LPS instillation, and in the group that had been both sensitized and challenged with OVA.

AB-PAS Histochemistry

The ratios of the four types of mucosubstance in goblet cells after LPS or OVA instillation (neutral glycoprotein, sialidase-sensitive sialomucin, sialidase-resistant sialomucin, and sulfomucin) are given in Figure 2. Because goblet cells were rarely seen in control rats, and there was no adequate amount of control mucosubstance in the septal epithelium, we examined the mucosubstance of goblet cells in the nasal opening of the incisive duct at the base of the nasal septum in the same section, where numerous goblet cells were present even in control rats. The incisive duct communicates with the oral cavity and also connects with the vomeronasal duct, functioning dually to conduct oral food orders to the olfactory epithelium and pheromones to the vomeronasal system.

In saline-instilled controls, most of the mucosubstance (75.5 ± 7.0% of total) was neutral glycoprotein, and sulfomucin occupied only 13.2 ± 4.5% of total. The newly produced mucosubstance in nasal septal epithelium after LPS instillation or OVA challenge contained higher amounts of sulfomucin and lower amounts of neutral glycoprotein (p < 0.01); LPS-induced mucosubstance contained more sulfomucin (70.1 ± 13.5% of total) and less neutral glycoprotein (8.6 ± 5.9%) than OVA-induced mucosubstance (sulfomucin, 33.6 ± 12.3%; neutral glycoprotein, 41.8 ± 5.1%; p < 0.01).

Lectin Histochemistry

Six lectins were studied: Arachis hypogaea (PNA), Maackia amurensis II (MAL-II), Sambucus nigra (SNA), Ulex europaeus I (UEA I), Dolichos biflorus (DBA), and Pisum sativum (PSA). Some of the mucosubstance in rat nasal epithelium reacted with four of the lectins: PNA, MAL-II, SNA, and UEA-I. This indicates the presence of galactose-N-acetylgalactosamine, α2,3- and α2,6-linked sialic acid–galactose, and fucose residues, respectively, in epithelial mucosubstance. The percent areas of lectin-stained mucosubstance in goblet cells are given in Figures 3 and 4. In every case, control slides that were incubated either without lectin or without ABC reagent, or with the lectin together with its inhibitory sugar, showed low levels of background staining.

PNA stained a small amount of the mucosubstance of goblet cells after saline instillation (3.5 ± 2.3% of total) or OVA challenge (5.8 ± 1.4%). LPS instillation induced a significant increase in PNA-reactive mucosubstance in nasal septal epithelium (64.8 ± 18.2%; p < 0.01).

The amount of MAL-II-reactive mucosubstance also increased significantly after LPS instillation (75.8 ± 22.1% of total), whereas 19.1 ± 6.1 and 32.3 ± 7.8% of the mucosubstance reacted with MAL-II after saline instillation or OVA challenge, respectively (p < 0.05).

UEA-I reacted with most of the mucosubstance, and SNA stained about half of the mucosubstance in goblet cells of rat nasal epithelium. The ratio of UEA-I or SNA-reactive mucosubstance did not change after LPS instillation or OVA challenge.

These results indicate the high amounts of fucose and α2,6-linked sialic acid–galactose residues, and the low amounts of galactose-N-acetylgalactosamine and α2,3-linked sialic acid– galactose residues, in the mucosubstance of rat nasal epithelium. LPS instillation induced hyperproduction of mucosubstance that contained more galactose-N-acetylgalactosamine and α2,3-linked sialic acid–galactose residues.

Rat Muc5ac mRNA Expression

Changes in rat Muc5ac gene expression in nasal septal epithelium were evaluated by reverse transcriptase (RT)-PCR. Both LPS instillation and OVA challenge significantly increased rat Muc5ac mRNA expression compared with animals treated with saline instillation (p < 0.05). However, there was no difference between LPS instillation and OVA challenge (Figure 5).

In the present study, hypertrophic and metaplastic changes of goblet cells were produced in rat nasal epithelium by intranasal challenge of OVA-sensitized allergic rats with OVA or intranasal LPS instillation. A similar increase in epithelial mucosubstance occurred 24 h after 3 d of LPS or OVA instillation. The newly produced mucosubstance contained more acid glycoprotein (sulfomucin) and less neutral glycoprotein in both groups. These results are consistent with other studies reporting the characteristics of respiratory mucus in hypersecretory states. Mucin differs in the sugar groups and the extent of sialylation and sulfation of the oligosaccharide structure. Increases in acid glycoproteins, especially sulfomucin, have been demonstrated in respiratory mucus from patients with chronic sinusitis (11), bronchial asthma (12), chronic bronchitis, and cystic fibrosis (13, 14). In animal experiments, tracheobronchial mucus contained more acid glycoproteins after antigen stimulation of allergic guinea pigs (15). Human neutrophil elastase induced acidic mucous secretion from hamster bronchial epithelial cells (16). There is a great diversity of terminal sugar groups in human respiratory mucus in different hypersecretory conditions (5, 6, 13, 14, 17). The alterations of carbohydrate structure have also been reported in pilocarpine- or antigen-induced secretions of sheep trachea (18, 19).

This is the first report showing the different properties of mucus produced by different inflammatory stimuli—allergic inflammation and LPS stimulation—in the same animal model. The comparative studies of newly produced mucosubstance revealed that there were differences in carbohydrate composition: (1) LPS-induced mucosubstance contained more sulfomucin and less neutral glycoprotein compared with OVA-induced mucosubstance; and (2) there were marked increases in galactose-N-acetylgalactosamine and α2,3-linked sialic acid–galactose residues in mucosubstance produced by LPS instillation.

Airway mucus serves a host defense function by entrapping and expelling inhaled bacteria and viruses on the mucociliary escalator. Carbohydrate structures of mucin are possible sites of attachment for pathogenic microorganisms. Several recognition sites have been described for respiratory pathogens, including Pseudomonas aeruginosa, Pseudomonas cepatia, Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenza, Mycoplasma pneumoniae, and influenza viruses (5, 20). In the present study, galactose-N-acetylgalactosamine and α2,3-linked sialic acid–galactose residues increased after LPS instillation. Galactose-N-acetylgalactosamine or α2,3-linked sialic acid–galactose residues were reported to be potential sites for the attachment of all these microorganisms, promoting their entrapment and removal by mucociliary clearance (21-23). The altered mucus may play an important role in the defense against bacterial infections, and it is interesting that these alterations did not occur in allergic inflammation in our study.

Sulfation of mucins commonly occurs in chronic airway diseases, including cystic fibrosis (CF); however, the role of sulfation in host defense against bacterial infection is still controversial. It has been reported that P. aeruginosa did not bind to sulfomucin, but had an affinity for sialomucin and neutral glycoprotein (14). Although Pseudomonas infection is characteristic of cystic fibrosis, mucus from patients with cystic fibrosis has reduced adhesion compared with mucus from patients with chronic bronchitis (14). If bacterial binding to stagnant mucus is a pathogenic mechanism in cystic fibrosis, sulfation of mucins may have the advantage of reducing bacterial infections.

Increased sulfation of mucus may be more important for airway inflammation because of the increase in negative charge. Mucin, the most plentiful high molecular weight polyanion on the respiratory surface, has been shown to interact with cationic inflammatory proteins, such as human leukocyte elastase, secretory leukocyte elastase inhibitor, and lysozyme (24, 25). The negatively charged sulfated carbohydrates are responsible for the inhibitory effect of mucins against these enzymes (26). In the present study, both allergic inflammation and LPS stimulation induced sulfation of mucins, although LPS-induced mucosubstance contained more sulfomucin. The negatively charged sulfomucin may play a role in protecting the mucosa against proteolysis by these cationic proteins or by bacterial enzymes both in allergic inflammation and in bacterial infection.

The diversity of mucus also results from the variety of mucin peptides encoded by mucin genes. Variable expression of mucin genes may lead to heterogeneous mucus in hypersecretory conditions. In the present study, both LPS instillation and OVA challenge significantly increased rat Muc5ac gene expression, and there was no difference between LPS instillation and OVA challenge. The result does not support our hypothesis that mucin genes are differentially expressed in various hypersecretory conditions, such as allergic inflammation and bacterial infection. Posttranslational modifications (glycosylation and sulfation), occurring mostly in the Golgi apparatus, may be more important for the disease-related alterations of mucus. However, it is possible that other, unknown mucin genes are responsible for the heterogeneity of mucus.

Specific inflammatory mediators and infiltrating cells are responsible for the hypersecretion of mucus induced by LPS stimulation or allergic inflammation. In a previous study, LPS-induced production of mucus was significantly inhibited by anti-rat neutrophil antiserum, neutrophil elastase inhibitor, indomethacin, and dexamethasone, whereas antigen-induced change was significantly inhibited by cysteinyl leukotriene (LT) antagonist and dexamethasone, and eosinophil infiltration did not relate to production of mucus (6, 7, 27). These results indicate that neutrophils, especially neutrophil elastase, and cyclooxygenase products may play an important role in LPS-induced production of mucus, and that cysteinyl-LTs are important for antigen-induced change. These specific mediators may also be responsible for the induction of specific glycosyltransferases in different hypersecretory conditions.

Respiratory mucus is heterogeneous in its carbohydrate structures. The alterations of carbohydrate chains of mucus in airway inflammation may be an important event for host defense, causing the entrapment of microorganisms or the neutralization of proteolytic enzymes; however, the biological functions of mucin carbohydrate chains are still unclear. It is hoped that such studies will improve the understanding and treatment of mucous hypersecretion in airway diseases.

The authors thank Ms. Chieko Shinohara for help in preparation of the figures.

Supported in part by a Grant-in-Aid for General Scientific Research from the Ministry of Education of Japan (No. 10470355), and by a Grant-in-Aid (1999) from the Mie Medical Research Foundation.

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Correspondence and requests for reprints should be addressed to Takeshi Shimizu, M.D., Department of Otorhinolaryngology, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514, Japan. E-mail:

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