Methylxanthines induce bronchospasmolytic effects in asthmatic patients due to their nonspecific inhibition of phosphodiesterases and a subsequent increase in intracellular levels of the second messenger cAMP. Methylxanthines also increase ciliary beat frequency and stimulate mucociliary transport. Previous studies have demonstrated overstimulation and damage to tracheal goblet cells as well as a substantial decrease in the proportion of goblet cells containing sialylated glycoconjugates after intravenous administration of aminophylline.

Fucosylated glycoconjugates have been identified by in both secretory granules and plasma membranes of cells in the respiratory tract. Glycoconjugates are components of secreted mucus and contribute substantially to viscoelastic properties of mucus. Both secreted glycoconjugates and those bound to cellular surfaces also serve as adhesion sites for antigens. An increased proportion of fucosylated glycoconjugates in the mucus of the respiratory tract has been described in sinusitis, chronic bronchitis, asthma, cystic fibrosis, and acute bronchiolitis in rats. In healthy adult rats, the (1-2) linkage of fucose to galactose is the most common linkage. Fucosylation in other linkages, (1-3), (1-4), and (1-6), respectively, to N-acetyl glucosamine increases under pathological conditions such as inflammation or cystic fibrosis at the expense of fucosylation in the (1-2) position and of sialylation.

Since the administration of aminophylline significantly lowers the proportion of sialylated glycoconjugate-containing tracheal goblet cells, we have evaluated the proportion of fucosylated glycoconjugate-containing goblet cells in the present study.

Sixteen SPF New Zealand White male rabbits (Charles River, Sulzfeld, Germany; mean body weight 2.31±0.38 kg) were used in the study. Six rabbits were given intravenous injections of the methylxanthine bronchodilator drug aminophylline (Syntophyllin inj.; Hoechst-Biotika, Martin, Slovakia; 5 mg/kg/body weight), and six rabbits were administered intravenously the methylxanthine vasodilator drug containing 4 mg etophylline and 1 mg theophylline per kg/body weight (Oxantil inj.; Hoechst-Biotika). The dose of Syntophyllin was chosen because it is the recommended dose for intravenous treatment of acute asthmatic attacks and this was the rationale for the selected dose of Oxantil as well. The material was collected at 15 and 30 min after treatment. Four untreated healthy rabbits served as controls. The specimens were collected immediately after the induction of anaesthesia.

The middle portions of tracheae between the 15th and 20th tracheal rings were formalin-fixed, paraffin-embedded, and sections of 5–7 ?m thickness were cut. Combined staining with alcian blue at pH 2.5 and PAS was used to demonstrate total acidic and neutral glycoconjugates and thus the total number of goblet cells. Lectin histochemical methods were used to detect fucosylated glycoconjugates. The legume lectin Ulex europaeus agglutinin I (ULE-I), detecting terminal or branched fucose (1-2) linked to an oligosaccharide, and the ascomycete orange-peel mushroom Aleuria aurantia lectin (AAL), detecting (1-6)-, (1-3)-, and (1-4)-linked fucose residues, were employed (Vector Laboratories, Burlingame, CA, USA). After dewaxing and rehydrating, endogenous peroxidase was blocked and the sections were incubated with either biotinylated ULE-I or biotinylated AAL alone or in combination with concentrations of 30 ?g/ml for 60 min at room temperature. The sections were then incubated with a solution of streptavidin–horseradish peroxidase conjugate (Vector Laboratories) in a concentration of 2 ?g/ml for 45 min, followed by a visualisation step using FAST™ DAB Peroxidase Substrate Tablets (Sigma-Aldrich Chemie, Deisenhofen, Germany) in combination with CuSO₄ to enhance staining. The blocking of endogenous peroxidase was verified by omitting the first step of the method. Specific lectin binding was verified by a 15-min preincubation of lectins with control substrate 0.2 M -fucose, preceding the incubation of sections.

Only goblet cells that were stained and contained well-developed granules were evaluated. The granules had to occupy at least 2/3 of a cell. Simultaneous application of both lectins enabled identification of goblet cells containing glycoconjugates that were positive for both lectins; therefore, the overlap of AAL- and ULE-I-positive goblet cells could be calculated. Venn’s diagrams enabled the calculation of goblet cells containing ULE-I-positive granules only and goblet cells containing AAL-positive granules only.

For statistical evaluation, numbers of goblet cells in five categories (all goblet cells, ULE-I-positive goblet cells, AAL-positive goblet cells, all lectin-positive goblet cells, and ULE-I- and AAL-positive goblet cells) were evaluated by the chi-square test of homogeneity in frequency tables, using Yates’ correction for low frequencies and the McNemar test of symmetry, respectively, when appropriate. These data were also evaluated by one-way ANOVA with the Kruskal–Wallis post hoc test and Levene’s test for equal variances followed by the Aspin-Welch test, when appropriate (Statistica v.6.0 software; StatSoft, Tulsa, OK, USA). The significance of differences between fucosylated glycoconjugate-detecting methods within individual groups was tested with the matched t-test, Spearman rank correlation, the matched sign test, and the Wilcoxon’s paired test (BMDP New System software; Statistical Solutions, Saugus, MA, USA).

The experimental procedures were performed under general anaesthesia (ketamine 35 mg/kg and xylazine 5 mg/kg, intramuscularly) and after the local subcutaneous infiltration of the ventral cervical field with procaine. The experimental procedure was approved by the Animals Protection Expert Commission of the Faculty.

The tracheae of both control and treated rabbits were lined with pseudostratified columnar ciliated epithelium largely composed of ciliated cells, goblet cells, and basal cells. The height of the epithelium was approximately 25–30 ?m. The distribution pattern of secretory elements was irregular. By using conventional histochemical methods, the secretory elements revealed typical staining patterns according to the type of glycoconjugates they contained. PAS-positive mucous granules were stained magenta; alcian blue-stained mucous granules were stained blue. Some goblet cells were stained in various shades of violet; these cells were considered to contain mixtures of acidic and neutral glycoconjugates. The appearance of goblet cells stained with lectins was identical in control and treated rabbits. Staining with ULE-1 resulted in stained mucous granules in goblet cells, either as a homogeneous content of granules, or as darkly-stained rings; staining of the zone of cilia occurred only in the close vicinity of apical surfaces of goblet cells. AAL staining resulted in positive individual mucous granules as dark rings. The zone of the cilia was always densely stained.

Figure 1. ULE-I staining of mucous granules in a goblet cell in tracheal epithelium of a rabbit at 15 min after intravenous administration of Oxantil. Note that a goblet cell contains unstained mucous granules (open arrow) in close vicinity of a goblet cell containing stained granules. Bar, 20 ?m.

Figure 2. AAL staining of mucous granules in a goblet cell (arrow) in tracheal epithelium of a rabbit at 15 min after intravenous administration of Oxantil. Bar, 20 ?m.

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