Persistent mucin glycoprotein alterations in equine recurrent airway obstruction

A. M. Jefcoat1, J. A. Hotchkiss2, V. Gerber1, J. R. Harkema2, C. B. Basbaum3, and N. E. Robinson1

1 Department of Large Animal Clinical Sciences and 2 Department of Veterinary Pathology, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824; and 3 Department of Anatomy, School of Medicine, University of California, San Francisco, California 94143


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (alpha -1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of alpha -1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.

bronchoalveolar lavage fluid; O-linked mucin glycoproteins; oligosaccharide side chains; lectins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACCUMULATION OF EXCESS MUCUS in airway lumens is a clinical feature of human airway diseases such as asthma, cystic fibrosis, and chronic bronchitis and of recurrent airway obstruction (RAO) in horses (10, 18, 22). RAO is an asthmalike condition of mature horses that is characterized by bronchoconstriction, airway wall thickening, and increased airway secretions occurring as a result of exposure to a dust, an allergen, and an endotoxin-laden indoor (barn) environment (18). Clinical signs are episodic, but the condition is progressive and permanent. Disease remission follows separation from the precipitating environment, but clinical signs recur on reexposure.

An increased mucus presence in airway lumens can be a direct cause of bronchial obstruction and can effectively increase resting airway wall thickness. This latter effect can amplify the lumen-narrowing effect of bronchoconstriction (15). Also, although RAO is not considered to be of infectious origin (16), it has been recognized in humans that mucus hypersecretion and/or decreased mucus clearance from the tracheobronchial tree can potentially predispose individuals to secondary bacterial colonization (10).

The mucus blanket that overlies the airway epithelium is composed of a liquid sol layer that surrounds the cilia and a more viscous gel layer above the sol (24). The gel layer is a complex mixture of water, electrolytes, lysozymes, cells, and mucin glycoproteins (10, 22, 24). Mucins are high molecular weight glycoproteins (154,000 to >7,000,000) that impart viscoelastic properties to mucus. They are composed of a core protein (the mucin apoprotein) to which numerous linear and branching oligosaccharide side chains are attached by means of specific O-glycosidic linkages (19, 20, 24). These side chains are composed of various combinations of five different individual sugar types: N-acetylgalactosamine, N-acetylglucosamine, galactose, sialic acid (N-acetylneuraminic acid), and fucose (4, 23). The addition of each sugar is dependent on the presence of a specific glycosyltransferase enzyme (23). Individual sugars in an oligosaccharide side chain can be linked to the preceding sugar molecule in a variety of ways; for example, a 1,2 linkage is a bond between the number 1 carbon of the distal sugar and the number 2 carbon of the proximal sugar, whereas a 1,3 linkage is between the distal carbon 1 and the proximal carbon 3.

The mucin oligosaccharide side chain structure can have functional importance, imparting specific binding activity toward structures such as bacterial adhesin molecules (20, 21) and potentially contributing to the degree of viscoelasticity of the mucous layer. As an example of the latter, fucose concentration has been positively correlated with mucus viscoelasticity (11). Individual mucin sugars such as fucose have also been used as markers of tracheobronchial mucus production in humans (9).

The purpose of this study was to develop methods for comparing mucus airway secretions in bronchoalveolar lavage fluid (BALF) of RAO-affected and control horses. This is a critical initial step in studying the mechanisms of altered mucus production during airway disease and accurately assessing the time course and functional significance of any accumulation. Although visual scoring systems of mucus quantity and quality have been previously employed (7, 14), a goal of this study was to use a more objective method to consistently analyze mucus secretion in the pulmonary airways. This report describes 1) the development and use of an enzyme-linked immunosorbent assay (ELISA) as an immunochemical method to quantify airway mucus secretions in horses and 2) the application of ELISAs and carbohydrate-specific lectin assays to the BALF from control and RAO-affected animals to identify possible quantitative and/or qualitative differences in mucins in these two populations of horses.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of RAO-Affected and Control Horses

In this study, horses from an RAO-affected herd that is maintained at Michigan State University (East Lansing) were used. To enter this herd, horses must meet the following criteria: 1) clinical signs of RAO, including cough, increased respiratory sounds, and increased expiratory abdominal effort, are observed during housing and abate when the horses are kept at pasture where there is no exposure to dust found in hay and stables; 2) horses develop changes in lung function compatible with airway obstruction when stabled and fed hay [maximal change in pleural pressure (Delta Pplmax) during tidal breathing >15 cmH2O]; and 3) airway obstruction is reversible, in part, with atropine. Control horses, also maintained in university herds, had no known history of chronic airway disease and did not display any clinical signs characteristic of obstructive airway disease when stabled.

Development of Murine Antibodies

Previous work (3) resulted in the generation of a panel of murine monoclonal antibodies directed against sheep tracheal secretions. Fifteen of these antibodies were screened for immunoreactivity against stored and secreted equine mucosubstances.

Screening of Antibodies

Immunofluoresence studies. Frozen microdissected horse pulmonary airways were embedded in optimum cutting temperature compound (Miles, Elkhart, IN), snap-frozen in a liquid nitrogen-cooled isopentane slush, and stored at -80°C. Sections were fixed in zinc-formalin for 5 min, washed two times with phosphate-buffered saline (PBS; pH 7.4), and blocked with 2% normal horse serum-0.3% Triton X-100 (Sigma, St. Louis, MO) in PBS for 25 min to cover irrelevant binding sites. The primary antibodies were diluted in the 2% normal horse serum solution: 1:100 for ascites antibodies and 1:20 for antibodies harvested from the culture supernatant. The antibodies were applied to airway sections and incubated at 37°C for 90 min. After a wash with rinsing buffer (1:10 dilution of 10× automation buffer; Biomedia, Foster City, CA), biotinylated horse anti-mouse IgG (VECTASTAIN Elite ABC kit, Vector Laboratories, Burlingame, CA) diluted 1:200 in 2% normal horse serum solution was added to each slide, and the slides were incubated for 30 min at 37°C followed by a wash with automation buffer. Fluorescein-conjugated avidin D (Vector Laboratories) was diluted 1:250 in automation buffer, applied to the tissue sections, and incubated for 30 min at 37°C. The sections were then washed with PBS and examined with an Olympus microscope equipped for epifluorescence studies. Each section was evaluated for location and intensity of immunofluorescent staining.

ELISA. Three ascites-derived and two culture supernatant-derived monoclonal antibodies with significant immunoreactivity to mucous goblet cells (based on immunofluorescence results) were screened for reactivity to mixed horse BALF in an ELISA. Serial dilutions from 1:100 to 1:1,280 were used for ascites antibodies, whereas dilutions for supernatant antibodies were 1:20 to 1:2,650. The diluent and blocking agent was 2% horse serum-0.3% Triton X-100 in PBS. The BALF was treated with 0.1% dithiothreitol (DTT; Sigma), shaken for 20 min to disperse the mucins, and then centrifuged at 1,500 rpm for 12 min to remove the cells. The supernatant was collected and dialyzed against distilled water overnight at 4°C to remove DTT. For the ELISA, 100 µl of cell-free supernatant were added to the wells of Immulon-4 HBX 96-well plates (Dynex Technologies, Chantilly, VA) in duplicate and incubated overnight at 40°C to thoroughly fix the antigen to the bottom of the wells. One hundred microliters of blocking agent (2% normal horse serum-0.3% Triton X-100 in PBS) was then added to each well to cover irrelevant binding sites and incubated at 37°C for 25 min. The plates were washed four times with 1× automation buffer, and then 100 µl of each primary antibody appropriately diluted in the blocking solution were added to the wells in duplicate followed by incubation at 37°C for 90 min. Washes with automation buffer were repeated, and 100 µl of biotinylated secondary antibody (VECTASTAIN Elite ABC kit) diluted 1:200 in 1× automation buffer was added to each well. The plates were incubated at 37°C for 30 min and then washed four times with automation buffer. One hundred microliters of VECTASTAIN ABC reagent were added to each well followed by 30 min of incubation at 37°C. After four washes with automation buffer, 100 µl of chromagen (o-phenylenediamine in 0.05 M phosphate-citrate buffer at 0.4 mg/ml; Sigma) were applied to each well. Just before addition to the wells, 30% hydrogen peroxide was added to the chromagen at a dilution of 1:2,500. After 5 min, the plates were read with a spectrophotometer at 450 nm, which gave optical density (OD) readings that corresponded to antigen levels in the samples (colorimetric assay).

Antibody Immunoreactivity With Purified Standard Mucins

Purified mucins from porcine stomach and bovine salivary gland (Sigma) were diluted with carbonate binding buffer (pH 9.5) at 10 µg/100 µl, and ELISA was performed with identical methods as described in ELISA.

Sodium Periodate Incubation

Porcine gastric mucin in carbonate binding buffer (10 µg/100 µl) and lavage samples from four RAO-affected horses were compared by ELISA before and after sodium periodate treatment. Sodium periodate oxidation attacks vicinyl groups of the sugars of glycoproteins, cleaving O-glycosidic linkages to the protein (2). Samples were dried to the bottom of Immulon-4 HBX 96-well plates at 40°C and then incubated with 2% horse serum blocking buffer (as described in ELISA) at 37°C for 90 min. One hundred microliters of 100 mM sodium periodate in 50 mM sodium acetate were added to the appropriate wells in duplicate. For comparison, untreated control wells (sample only) and wells with sample, sodium periodate, and glucose were utilized [glucose blocks the action of sodium periodate (2)]. For wells with added glucose, 100 µl of 100 mM sodium periodate-0.1 M glucose in 50 mM sodium acetate were used. The plates were then incubated at room temperature overnight (in the dark). After the overnight incubation, sodium periodate and sodium periodate-glucose wells were incubated for 30 min at room temperature with 10 mM sodium borohydride to prevent nonspecific cross-linking of antigen to antibody by Schiff base formation. The plates were washed four times with 1× automation buffer, and then ELISA was performed as described in ELISA.

Chondroitinase ABC and Heparinase Incubation

ELISA comparison of BALF before and after incubation with the proteoglycan-degrading enzymes chondroitinase ABC and heparinase was performed to differentiate antibody-mucin glycoprotein binding from antibody binding to O-linked glycosaminoglycan epitopes. The samples were the same as those used for the sodium periodate incubation. After the antigen was fixed to the wells and the nonspecific binding sites were blocked, the samples were incubated overnight with chondroitinase ABC (50 µU/well in 0.05 M Tris, pH 8.0) or heparinase (250 µU/well in 0.05 M Tris, pH 6.8, with 5 mM calcium chloride) at 37°C. After incubation, the plates were washed four times with 1× automation buffer in preparation for ELISA, and a colorimetric enzyme-linked assay was performed.

ELISA of Serum From Control and RAO-Affected Horses

Colorimetric ELISA was performed on the serum from 10 control and 10 RAO-affected horses to test for the presence of non-airway-specific immunoreactive antigen in both groups of horses.

ELISA of Native Tracheal Mucus

Mucus was harvested directly from the trachea of an RAO-affected horse at necropsy. The mucus was weighed, diluted to 10% in PBS, and then applied to microtiter plates for colorimetric 4E4-based ELISA.

High Molecular Weight Cutoff Dialysis

BALF was collected from four RAO-affected horses and prepared as described in ELISA. The BALF was then dialyzed overnight at 4°C against distilled milliQ water in 100,000 molecular weight cutoff (MWCO) dialysis tubing (Spectra/Por, Spectrum Medical Industries, Houston, TX). ELISA was then performed on milliQ water and nondialyzed BALF as controls and on all dialyzed samples and dialysates. The dialysate was tested in both native and 5× concentrated forms. Concentrations were made with 30,000 MWCO centrifugal concentrators (Centriprep, Amicon, Beverly, MA). For ELISAs, the samples were plated (100 µl/well in triplicate), incubated overnight, and blocked with an 8% purified casein solution (Roche Diagnostics, Indianapolis, IN). After secondary antibody and ABC reagent incubations, with dilutions and volumes identical to those described in ELISA, 100 µl of QuantaBlu fluorogenic peroxidase substrate (Pierce, Rockford, IL) were added to each well, and the plates were read at 5-min intervals for 20 min (kinetic runs) with a SpectraMax Gemini fluorescent plate reader (Molecular Devices, Sunnyvale, CA), which detected the amount of fluorescence emitted from the reaction (in relative fluorescence units). The maximum slope of the kinetic display of relative fluorescence units versus time was calculated with SOFTmax PRO software (Molecular Devices) and is reported as Vmax units per second. Vmax units per second values were then used as end points for sample comparisons (fluorogenic assay), with higher values corresponding to increased presence of target molecule.

ELISA of BALF From Control and RAO-Affected Horses

BALF was collected from 6 control (5 being 7 yr of age or younger, 1 being 12 yr of age) and 6 RAO-affected horses (10+ yr of age). Control horses were sampled after 48 h of indoor housing (exposure). The BALF from RAO-affected horses was collected and tested after the horses were stabled for 48 h (exposure) and after pasture for 30 days (pasture). The BALF was collected by means of a 3-m lavage tube (8-mm external diameter; Bivona, Gary, IN) wedged in a peripheral bronchus. Three 100-ml aliquots of sterile PBS were infused and recovered by suction after each 100-ml infusion, and the samples were pooled. Total and differential white blood cell counts were performed on all BALF. Total cell counts were performed by use of a hemacytometer within 2 h of collection. For differential counts, slides prepared with a Cytospin 3 centrifuge (Shandon, Pittsburgh, PA) were stained with Diff-Quik (Baxter Health Care, Dade Division, Miami, FL). With the exception of aliquots taken for cell counts, the samples were treated with 0.1% DTT and centrifuged at 1,500 rpm for 12 min to remove the cells. The supernatant was collected and dialyzed overnight at 4°C. ELISAs were performed with fluorogenic peroxidase substrate and a fluorescent plate reader as described in ELISA. Negative control well values (background) were subtracted from all sample readings.

Enzyme-Linked Lectin Assay

Lectins are well recognized as specific carbohydrate-binding molecules. Lectins for each of the five sugar types found in O-linked mucins (Table 1) were used in enzyme-linked lectin assays (ELLAs). BALF samples from RAO-affected horses at pasture and after 48 h of housing were compared with BALF samples from control horses after 48 h of exposure. BALF samples were incubated overnight in the same manner as described for ELISAs. The wells were blocked for 35 min with 8% casein blocking reagent. After being blocked, the wells were washed three times with 1× automation buffer. One hundred microliters of biotinylated lectins specific for alpha -1,2-fucose [Ulex europaeus agglutinin (UEA) I; 0.5 µg/ml], N-acetylglucosamine [succinylated wheat germ agglutinin (WGA); 0.5 µg/ml], N-acetylgalactosamine [soybean agglutinin (SBA; Glycine max); 1.0 µg/ml], sialic acid [Maackia amurensis lectin (MAL) II; 0.75 µg/ml PBS], and galactose [Ricinus communis agglutinin (RCA) I; 0.5 µg/ml PBS] were then added to each well and incubated for 45 min at 37°C (all lectins from Vector Laboratories). Diluent for each lectin was per supplier recommendation. The plates were then washed four times with 1× automation buffer. ABC reagent and QuantaBlu were added as described in High Molecular Weight Cutoff Dialysis, and fluorescence (in Vmax units/s) was measured. As with the ELISA, negative control well values (background) were subtracted from all sample readings.

                              
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Table 1.   Lectins used in enzyme-linked assays

Lectin assays with UEA 1, succinylated WGA, and MAL II were also performed on serum collected from two control horses.

BALF Protein Content

BALF samples were analyzed for protein content with the bicinchoninic acid method. Ten microliters of bovine serum albumin standard (serially diluted 1:2 from 1,000 to 8.44 µg/ml) and each sample were plated in duplicate on a 96-well microtiter plate. Two hundred microliters of bicinchoninic acid working reagent (Pierce, Rockford, IL) were added to each well. The plate was shaken for 30 s and incubated at 37°C for 30 min. The plate was then cooled to room temperature, and absorbance was measured at 550 nm.

Lung Function Testing

For confirmation of chronic RAO in disease-affected animals, Delta Pplmax was recorded during the indoor housing period and after time at pasture. The change in pleural pressure (Delta Ppl) was measured by use of an esophageal balloon connected to a pressure transducer (Validyne model DP45-22) system. Calculations were performed with a lung function computer (Buxco, Sharon, CT) (1) from 30 breaths (17).

ELLA and High MWCO Dialysis

BALF samples from four RAO-affected horses were dialyzed in the same manner as for high MWCO ELISA. ELLA procedures with UEA I, succinylated WGA, and MAL II were performed on samples before and after dialysis.

Mucin Standard Serial Dilutions

All ELISA and ELLA plates contained a serial dilution of porcine gastric and bovine salivary gland mucins (from 100 to 0.001 µg/100 µl) diluted in PBS as a means to quantify the amount of mucin in samples and standardize multiple plates. Mucin standards were treated in an identical manner as BALF samples.

Statistical Analyses

For direct comparisons of one factor between two groups, means of groups were compared with Student's t-test. For comparisons of one factor within a group, a paired t-test was employed. Multiple comparisons between groups were performed with one-way ANOVA. Tukey's test was used for post hoc analysis. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Confirmation of RAO-Affected Population

All RAO-affected horses displayed clinical signs of obstructive pulmonary disease during housing, with Delta Ppl exceeding 15 cmH2O (mean ± SE = 59.4 ± 9.8 cmH2O). The mean value on day 30 (pasture) was 15.1 ± 2.7 (SE) cmH2O. The control horses did not display any signs of obstructive pulmonary disease during 48 h of indoor housing. BALF cytology from the horses used for the ELISA and ELLA experiments is presented in Table 2. Both total cell counts and percent neutrophils were increased in RAO-affected horses after the housing period compared with pasture and control values.

                              
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Table 2.   Bronchoalveolar lavage cytology from control and RAO-affected horses

Screening of Antibodies

Immunofluoresence. Five of fifteen antibodies bound strongly to secretory cells in the surface epithelium and submucosal glands of airway sections. These antibodies, 4E4, 5C7C6, IG5C11F10, 3F11 and 3C10, were selected for use in pilot ELISA studies. Figure 1 is a photomicrograph of the binding activity of antibodies 5C7C6, 3F11, and 4E4.


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Fig. 1.   Photomicrographs of monoclonal antibody binding to horse airway epithelium (immunofluorescence). A: negative control (no antibody). B: antibody 3F11. C: antibody 5C7C6. D: antibody 4E4. Yellow-green fluorescence indicates binding of monoclonal antibodies. Sections were also stained with propidium homodimer to label nuclei. L, airway lumen; E, airway epithelium; LP, lamina propria. Arrowheads, mucosubstance in surface epithelium and submucosal glands.

ELISA on mixed BALF. Only antibodies 4E4, 5C7C6, and 3F11 displayed noticeable immunoreactivity with BALF (Fig. 2).


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Fig. 2.   Binding of indicated monoclonal antibodies (AB) to epitopes in horse bronchoalveolar lavage fluid. A: ascites-derived antibodies. B: culture supernatant-derived antibodies. OD, optical density. Values are means ± SD of duplicate wells. Bars not seen are within symbol.

As shown in Fig. 2, ELISA-negative control values were near zero, and the highest value generated at the greatest antibody concentration was an OD of 2.0. Therefore, an OD of 1.0 at 450 nm was arbitrarily chosen as a standard reference value to quantify secretory product in BALF with a colorimetric assay. Dilutions of antibodies 4E4, 5C7C6, and 3F11 that corresponded with an approximate OD of 1.0 (1:400, 1:100 and 1:80, respectively) were then chosen for future studies.

ELISA on Standard Mucins

Antibodies 4E4, 5C7C6, and 3F11 all generated OD values of >1.0 when applied to beef salivary gland mucin. 5C7C6 and 3F11 displayed similar low levels of reactivity toward porcine gastric mucin as toward bovine mucin, but 4E4 exhibited strong immunoreactivity to porcine mucin (Fig. 3).


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Fig. 3.   Monoclonal antibody binding to purified porcine gastric mucin. 1, 4E4; 2, 3F11; 3, 5C7C6; 4, negative control (no antibody). Values are means ± SD of 4 replicates. * 4E4 binding was significantly elevated over negative control, P < 0.05.

Sodium Periodate Incubation

With 4E4 as the primary antibody, a mean post-sodium periodate incubation OD value of 0.44 ± 0.08 (SD) between duplicate wells was significantly different from the mean preincubation OD value of 1.19 ± 0.15 (SD). The mean negative control well value (no antibody) was 0.132. Sodium periodate effects were blocked by the presence of 0.1 M glucose in glucose-added wells.

Chondroitinase ABC and Heparinase Incubation

Neither chondroitinase nor heparinase treatment affected 4E4 binding. The mean postincubation OD values of 1.33 ± 0.37 (SD) between duplicate wells for chondroitinase and 1.26 ± 0.35 (SD) for heparinase were not significantly different from the mean preincubation value of 1.24 ± 0.32 (SD).

Serum ELISA

4E4-immunoreactive antigen was not detected in the serum of either control or RAO-affected horses. The mean OD for 10 control horses was 0.197 ± 0.025 (SD), whereas the mean OD for 10 disease-affected horses was 0.191 ± 0.006 (SD). No significant differences existed between the two groups. Negative control values were 0.220 ± 0.009 (SD).

Tracheal Mucus ELISA

4E4-immunoreactive glycoprotein was detected in diluted directly harvested tracheal mucus. The amount of 4E4 binding in 10% tracheal mucus was similar to that of the porcine mucin standard at 10 µg/100 µl (OD values of 1.418 and 1.366, respectively).

High MWCO Dialysis

4E4-based ELISA showed no decrease in signal after BALF samples had been dialyzed in 100,000 MWCO tubing. The mean value for predialysis samples was 3.1074 ± 0.866 (SE) Vmax units/s, whereas the postdialysis value was 2.746 ± 0.464 (SE) Vmax units/s.

BALF ELISA for Control and RAO-Affected Horses

4E4-based ELISA showed a significantly elevated mean Vmax units per second value in the RAO-affected horses during exposure compared with the control value (Fig. 4A). The mean ELISA reading for RAO-affected horses in remission decreased from that in acute disease but remained greater than the control level, which was the near background level. The average volume of BALF returned and the total and differential cell counts for each group of horses are given in Table 2.


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Fig. 4.   Results of 4E4-based ELISAs and enzyme-linked lectin assays (ELLAs) for control and recurrent airway obstruction (RAO)-affected horses (n = 6/groups). 1, control horses; 2, RAO exposure; 3, RAO pasture. A: 4E4 ELISA. RAO-affected horses had significantly elevated 4E4-immunoreactive glycoprotein levels in bronchoalveolar lavage fluid (BALF) during exposure (acute disease) compared with control horses. B: Ulex europaeus agglutinin I (alpha -1,2-fucose-specific) ELLA. RAO-affected horses had significantly elevated alpha -1,2-fucose levels in BALF during pasture (remission) and exposure (acute disease) compared with control horses. C: succinylated wheat germ agglutinin [N-acetylglucosamine (GlcNAc)-specific] ELLA. RAO-affected horses had significantly elevated GlcNAc levels in BALF during exposure and at pasture compared with control horses. D: Maackia amurensis lectin II (sialic acid-specific) ELLA. Values are means ± SE. * P < 0.05.

ELLAs

Significant elevations were noted in alpha -1,2-fucose and N-acetylglucosamine levels in RAO-affected horses during exposure and at pasture compared with those in control horses (Fig. 4, B and C). There were also increased sialic acid levels in RAO-affected horses during exposure and at pasture compared with control levels, with a trend toward significant differences between groups (P = 0.074; Fig. 4D). Although the porcine mucin standard responded to soybean agglutinin, the detected level of N-acetylgalactosamine in all groups of horses was not distinguishable from the background level, and no significant differences existed. No significant differences in the levels of galactose existed between groups.

UEA I, succinylated WGA, and MAL II did not respond to serum elements. Mean Vmax values for the lectins were 0.319 ± 0.02 (SD), 0.336 ± 0.06, and 0.277 ± 0.10 Vmax units/s, respectively. None of these were elevated over background Vmax units per second values.

Protein Assay

Total protein in BALF from RAO-affected horses in acute disease was significantly greater than that in control horses. Protein levels in BALF from RAO-affected horses in remission remained elevated over control levels, but there was no significant difference. The mean absorbance value for RAO horses in acute disease was 0.394 ± 0.038 (SE), which corresponded to 800 µg/ml of protein. Absorbance values for control and RAO-affected horses in remission were 0.198 ± 0.025 and 0.290 ± 0.047, respectively, corresponding to 250 and 500 µg/ml of protein.

High MWCO Dialysis ELLAs

No decrease in signal compared with that in the predialyzed sample was noted in BALF after 100,000 MWCO dialysis for any of the tested lectins (Fig. 5).


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Fig. 5.   ELLAs of 4 BALF samples from RAO-affected horses before and after 100,000 molecular weight cutoff dialysis. 1, alpha -1,2-fucose; 2, N-acetylglucosamine; 3, sialic acid. Values are means ± SE. No decrease in signal indicated that lectin-specific sugars are components of molecules > 100,000.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The experimental findings detailed in this study are the results of the first study, to our knowledge, to utilize monoclonal antibodies and lectins to examine disease-associated changes in glycoprotein secretion in RAO-affected horses during exacerbation and remission. Novel investigation regarding airway secretion in RAO can be of benefit on two fronts because RAO is both a significant disease of the horse population and a condition with similarities to chronic human airway diseases such as asthma.

Confirmation of Study Populations

Inclusion of individual horses into either control or RAO-affected populations was determined by three factors accepted as criteria for establishing a phenotype of RAO (16): presence of clinical signs, BALF cytology data, and lung function testing. Clinical signs of obstructive airway disease were observed in RAO-affected but not in control horses after indoor housing. RAO-affected horses showed significant increases in both total cell counts and percent neutrophils in BALF during indoor housing compared with pasture or to control horses. This pattern is characteristic of RAO (6, 12). Lung function test results showed that all RAO-affected horses had Delta Pplmax values of >15 cmH2O after 48 h of indoor housing, paralleling development of clinical signs of obstructive disease and increases in total cells and percent neutrophils in BALF. This pattern of an elevated Delta Ppl in response to natural challenge is typical of RAO (13).

Monoclonal Antibody 4E4 as a Tool for Identification and Quantification of Secreted Mucous Cell Product

4E4 was chosen from a panel of monoclonal antibodies as a primary antibody for use in an ELISA that can be applied to BALF from horses to quantify mucus secretions. The recognition by 4E4 of a mucin or mucinlike molecule was evidenced by its binding to mucous cells in airway epithelium and its immunoreactivity to a large (>100,000) glycoprotein present in BALF. Antibody binding was not affected by chondroitinase or heparinase treatment, further indicating its specificity for a mucin or mucins. Additionally, a 4E4-recognized epitope was present on purified porcine gastric mucin molecules. As well as serving to help identify the target of the antibody, 4E4 immunoreactivity to a commercially available mucin allowed development of a standard ELISA reference curve.

4E4-Based ELISAs and ELLAs of BALF

Levels of a 4E4-immunoreactive molecule were significantly increased in the RAO-affected horses during indoor exposure compared with those in the control horses. Levels in RAO-affected horses decreased when the horses were in remission but remained elevated compared with the control levels, which were near background readings. Levels of sialic acid followed the same pattern as 4E4, although significant differences were not present (P = 0.074). These results indicated that 4E4 and, to a lesser degree, sialic acid measured an increase in mucus production in RAO-affected horses during acute disease and suggested that there is a persistent nature to altered mucus production in the disease state.

Results of enzyme-linked assays with alpha -1,2-fucose- and N-acetylglucosamine-specific lectins demonstrated disease-dependent increases in these sugars that persisted 30 days after the horses were removed from environmental challenge. Although mean values at 30 days were less than those observed during acute disease, a significant elevation remained. The pronounced neutrophilic inflammation in RAO-affected horses in acute disease markedly abated during clinical remission, but elevations in total protein and percent neutrophils did remain in diseased horses after 30 days at pasture compared with control levels. Inflammatory mediators such as neutrophil elastase have long been known to be mucus secretagogues (8). It is therefore possible that the persistently increased levels of mucin glycoproteins detected in BALF may be secondary to sustained, although attenuated, inflammation. Supporting this hypothesis is a recent study (5) describing persistent granulocyte-dependent activation of nuclear factor-kappa B in bronchial brushing samples from RAO-affected horses even after removal from challenge for 21 days. Although a link between sustained inflammation and increased mucin secretion can be hypothesized, the exact mechanisms of persistent glycosylation changes, such as upregulation of specific glycosyltransferases and mucin apoproteins, increased glycosyltransferase and apoprotein mRNA stability, or altered availability of mucin sugar molecules, have not been identified.

High MWCO dialysis experiments with multiple lectins yielded similar results as with the 4E4-based assay, where no loss of signal was detected in samples after dialysis in 100,000 MWCO tubing. This demonstrated that lectin-based differences detected in BALF were due to glycosylation patterns that are integral parts of larger molecules (i.e., mucin glycoproteins) and are not due to individual sugar or small soluble sugar-bearing molecules in airway fluids. In addition, lack of target molecules in serum indicated that serum leakage into the airways, as expected with airway inflammation, did not influence the test results.

The mean Delta Ppl value in the RAO-affected horses in remission, although significantly lower than the mean value in acute disease, remained >15 cmH2O, the minimum value accepted as an indicator of airway obstruction in RAO-affected horses postexposure (16). Although other factors such as airway wall thickening may be involved, maintenance of airway obstruction while at pasture coupled with persistent mucin alterations suggested that an increased or abnormal mucus presence in remission may have contributed to long-term low-level airway obstruction in the RAO-affected horses.

Although not measures of the absolute amounts of mucin glycoproteins or mucin carbohydrates in the airways, the ELISAs and ELLAs used in this study characterized mucin glycoprotein components and demonstrated significant relative differences in the levels of the target molecules in BALF from RAO-affected and control horses, highlighting disease and exposure effects on airway secretory products.

Collectively, these results indicated that disease-dependent alterations in the quantity and/or quality of mucin oligosaccharide side chains occurred in the horses with the asthmalike condition of RAO. Most significantly, persistent elevations in secreted mucin glycoproteins were evident in the airways of RAO-affected horses, perhaps secondary to sustained low-level inflammation.


    ACKNOWLEDGEMENTS

We thank Cathy Berney for technical assistance.


    FOOTNOTES

This study was supported by US Department of Agriculture Cooperative State Research, Education, and Extension Service Grant 99-35204-8366), National Center for Research Resources Grant F32-RR-05070, and the American Horse Shows Association.

Address for reprint requests and other correspondence: A. M. Jefcoat, 210 National Food Safety and Toxicology Center, Michigan State Univ., East Lansing, MI 48824-1302 (E-mail: jefcoata{at}pilot.msu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 October 2000; accepted in final form 7 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 281(3):L704-L712
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