Twelve healthy subjects and 13 subjects with asthma were studied (Table 1). The subjects with asthma were taking inhaled short-acting β agonists only as treatment of their asthma. All subjects signed consent forms approved by the Committee on Human Research at the University of California, San Francisco. The study involved three visits for spirometry, allergen skin testing, and methacholine challenge (Visit 1), as well as sputum induction (Visit 2, 48 h after Visit 1) and bronchoscopy (Visit 3, 7 d after Visit 2), and these tests were performed as previously described (17-19). Sputum induction was performed according a 12-min sputum induction protocol (18). During bronchoscopy, the bronchoscope was introduced orally and advanced to the right mainstem bronchus, where up to nine mucosal biopsies (six or seven for morphometry and immunohistochemistry and two or three for RNA extraction) were taken from the carinae of the upper lobe, middle lobe, and superior segment, using a spiked fenestrated forceps.

Induced sputum was homogenized by mixing with an equal volume of 0.1% dithiothreitol, as previously described (20). Secreted mucin was measured in supernatants of induced sputum by enzyme-linked immunosorbent assay (ELISA), as previously described (21). Briefly, Dynex (Chantilly, VA) Microtiter plates (Immulon 2HB) were coated with purified IgG 17Q2 (0.2 μg/well) in coating buffer (0.05 M sodium carbonate buffer, pH 9.6) and incubated at 37°C for 2 h under an airtight cover. After washing with a solution of phosphate-buffered saline (PBS)–Tween 20 (0.05%), various amounts of standard mucin in PBS–Tween 20 (0.05%) (0.5. 1, 2, 4, 8, and 16 ng of protein per well) and the samples were added to the wells. These reactions were carried out under the same airtight conditions at 37° C for 2 h. After washing with PBS–Tween 20 solution, plates were reacted with alkaline phosphatase-conjugated IgG 17Q2 (0.2 to 0.4 μg/well) in 0.2% albumin and PBS–Tween 20. Incubation was carried out at 37° C for 2 h. Finally, after washing with PBS–Tween 20, phosphate substrate solution ( p-nitrophenyl phosphate, disodium, at 1 mg/ml in 10% diethanolamine solution, pH 9.8) was added for color development. The reaction was stopped by the addition of 50 μl of 3 N NaOH to each well. Developed color in each well was read at a wavelength of 405 nm.

This double-sandwich ELISA method measures “general” airway mucin, that is, the 17Q2 antibody specifically stains the granules of the surface goblet cells and mucous gland cells (lesser intensity) and the high molecular weight mucous glycoproteins found in sputum. Therefore, this general airway mucin probably includes both MUC5B and MUC5AC, and other mucin gene products. The epitope characterization for the 17Q2 antibody has been published previously (21).

Biopsies were fixed in 4% paraformaldehyde, dehydrated in alcohol, and infiltrated with glycomethacrylate (Polysciences, Warrington, PA). A 2-μM section was cut from each block and stained with Alcian blue and periodic acid–Schiff reagent (Sigma, St. Louis, MO). A microscopic image of each biopsy at a final magnification of ×80 was captured by video camera (3CCD; Dage, Michigan City, IN) linked to a computer (Power Tower Pro 200; Power Computing, Austin, TX) and NIH Image software (version 1.60; NIH, Bethesda, MD). A design-based stereologic analysis was then performed with NIH Image, Stereology Toolbox (Morphometrix, Davis, CA), and the Computer-Assisted Stereology Toolbox software system (C.A.S.T-Grid; Olympus, Albertslund, Denmark). Random sampling of high-power fields of airway epithelium was achieved by labeling areas of intact epithelium with a line on the underlying basement membrane. Two to six images per section (minimum of two, maximum of six, depending on the length of the intact epithelium) at a magnification of ×1,200 were sampled randomly at equal distances apart on the basement membrane line. Adequate biopsies were those with at least two high-power fields of intact epithelium. The median number of adequate biopsies analyzed in both groups was five (range, three to seven).

The volume density of mucin in goblet cells in the airway epithelium and the surface area of the epithelial basal lamina were determined by point and intersection counting (cycloid grid, Stereology Toolbox) (Figure 2). The number of goblet cells per volume of epithelium was calculated as follows: Nv_gob,epi = Vv_gob,epi/V_gob, where Vv_gob,epi is the volume density of goblet cells in epithelium and V_gob is the number-weighted volume of the goblet cell (22). The number-weighted volume of 20 goblet cells per biopsy (randomly sampled) was measured with a rotator, a local volume estimator in the C.A.S.T-Grid system (23).

Fig. 2. The volume of stored mucin in goblet cells in the airway epithelium in 13 subjects with asthma and 12 healthy subjects. The volume density of mucin stored in goblet cells in the airway epithelium was determined by point counting, using a cycloid grid consisting of points and lines (Stereology Toolbox). Points within the grid falling on areas staining positive by Alcian blue and PAS were counted as points on mucin (Pt_muc). Points within the grid falling on epithelial cells of all cell types were counted as points on epithelium (Pt_epi). Lines within the grid intersecting the basal lamina were counted as lines intersecting the basal lamina (L_bl). The volume of mucin per volume of epithelium (Vv_muc,epi) was calculated as

$${\text{Vv}}_{\text{muc},\text{epi}}=\frac{\Sigma {\text{Pt}}_{\text{muc}}}{\Sigma {\text{Pt}}_{\text{epi}}}$$

The surface of basal lamina per unit volume of epithelium (Sv_bl,epi) was calculated as

$${\text{Sv}}_{\text{bl},\text{epi}}=\frac{2\Sigma L_{\text{bl}}}{{\text{Pt}}_{\text{epi}}(l/\text{Pt})}$$

where l/Pt is the length per test point. The volume of mucin per unit surface area of basement membrane (in mm³/mm²) was calculated as

$${\text{Vs}}_{\text{muc},\text{bl}}=\frac{{\text{Vv}}_{\text{muc},\text{epi}}}{{\text{Sv}}_{\text{bl},\text{epi}}}({\text{in mm}}^3/{\text{mm}}^2).$$

Download Figure | Download Powerpoint

[More] [Minimize]

The MUC5B-like antibody (11C1; diluted 1:100) was generated from a hybridoma in which the immunogen was a secretory product of primary human tracheobronchial epithelial cells, and the specificity of the antibody was demonstrated by ELISA and Western blot. 11C1 is an IgG1 monoclonal antibody specifically reactive to submucosal gland cells rather than surface epithelial cells. Western blot demonstrated the recognition of the same mucin molecule recognized by the 17Q2 antibody. However, ELISA suggested that the 11C1 activity is blocked by a synthetic peptide representing the naked region of human MUC5B. In addition, the epitope was sensitive to protease treatment, suggesting that the antibody recognizes the unglycosylated MUC5B peptide. The ELISA was carried out in two ways. One involved the use of a sputum mucin-coated plate for the 11C1 reaction; before the reaction, the antibody was preincubated with 5 μg of MUC5B peptide. The competition was ∼ 50%. The second ELISA approach used MUC5B peptide to coat the plate, which was then reacted with 11C1 antibody. 11C1 reacts with MUC5B in this solid phase whereas the 17Q2 antibody does not react. Subsequently, Western blots (in 1% agarose gel) were used and both 17Q2 and 11C1 recognized the same mucin species (as represented by smears in the very high molecular weight region). However, in additional Western blot experiments 17Q2 recognized protease-treated sputum mucin (purified from a CsCl gradient and two cycles of gel filtration of the void volume region), but 11C1 did not, indicating that 11C1 recognizes the naked peptide region of sputum mucin.

The MUC1 monoclonal antibody (1:50 dilution) was purchased from Neomarkers (clone GP1.4; Neomarkers, Fremont, CA); data from the manufacturer indicate that this IgG1 antibody was raised against human milk fat globule membranes and is produced by a fusion between BALB/c splenocytes and mouse myeloma P3-X63-Ag8.653 cells. In Western blotting it recognizes two glycoproteins in the molecular weight range of 265K–400K, identified as the epithelial membrane antigen, or mucin 1 (MUC1). MUC1 antibody reacts with the DTRP epitope in the tandem repeats.

The MUC2 monoclonal antibody (1:50 dilution) was also purchased from Neomarkers (clone CCP58); data from the manufacturer indicate that this IgG1 antbody was raised against a synthetic peptide of 29 amino acids (KYPTTTPISTTTMVTPTPTPTGTQTPTTT) from MUC2 protein, coupled to keyhole limpet hemocyanin (KLH). It is produced by a fusion between BALB/c splenocytes and mouse p3-NS-1/Ag4-1 (NS1) cells. It recognizes a single protein of 520 kDa, identified as MUC2 glycoprotein. For MUC2 an additional rabbit polyclonal antibody was used (anti-MRP, 1:50 dilution, courtesy of J. Gum, UCSF).

The MUC5AC monoclonal antibody (1:50 dilution) was also purchased from Neomarkers (clone 1-13M1); data from the manufacturer indicate that this IgG1 antbody was raised against mucin M1from the fluid of an ovarian mucinous cyst from an O Le(a-b-) patient. It is produced by a fusion between BALB/c splenocytes and mouse myeloma Sp2/0 cells. It recognizes the peptide core of gastric mucin M1.

For the immunohistochemistry experiments, preimmune horse serum and a mouse purified plasmacytoma IgG1 (1:50 dilution, MOPC-31c; Sigma) were used as controls. Antigen retrieval was achieved by trial incubations with untreated, trypsin-treated, or sodium citrate-treated sections. Sections for MUC5AC and MUC5B staining were incubated with trypsin (Zymed Laboratories, South San Francisco, CA) at a 1:1 dilution for 45 min at 37° C, whereas sections for MUC2 staining were boiled in 10 mM sodium citrate buffer, pH 6, for 15 min and a deglycosylation step with 1% periodic acid was also included in some experiments (24). No antigen retrieval step was used for MUC1 staining. Nonspecific binding was blocked by incubation in 1.5% horse serum. Primary antibody incubation was for 16–24 h, biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) incubation was for 2 h, avidin–peroxidase antibody complex (Vector Laboratories) incubation was for 2 h, diaminobenzidine (DAB) chromogen (Zymed Laboratories) incubation was for 10 min, and counterstaining with Gill's hematoxylin (Sigma) was for 15 s. The intensity of immunostaining was scored visually (0 to 5+ scale).

Biopsies were immersed in 20% sucrose at 4° C for 2–4 h and placed in O.C.T. compound (Sakura Finetek U.S.A., Torrance, CA) before being snap frozen in liquid nitrogen. A 5-μm section from each biopsy was stained with Gill's hematoxylin and biopsies with intact epithelium but lacking submucosal gland tissue were selected; 23 of the 25 subjects had biopsies with these criteria. Total cellular RNA was obtained from these biopsies by trimming off the O.C.T. compound, sonicating the biopsy in 0.5 ml of RNAzol (Tel-Test, Friendswood, TX), and isolating RNA according to the method of Chomczynski and Sacchi (25). RNA samples were rigorously tested using –RT (reverse transcriptase) controls in order to evaluate RNA contamination with DNA. In most cases the difference between –RT and +RT data points was within 10–20 threshold cycles (C_t, the threshold cycle in real-time polymerase chain reaction [PCR] that reflects mRNA copy numbers in a given RNA sample), indicating less than 0.1% contamination with genomic DNA. Four of 23 patient samples were judged to have excessive genomic DNA contamination. In samples from the remaining 8 healthy subjects and 11 subjects with asthma transferrin receptor delta C_t values (+RT versus –RT controls) were at least 10 and did not interfere with RNA analysis. Duplicate biopsies were available from 3 of the 8 healthy subjects and from 4 of the 11 subjects with asthma; these duplicates were also analyzed and averaged.

The expression of mucin genes was measured by using real-time quantitative PCR (26-28). Existing procedures were modified to improve the sensitivity of the assays and to allow simultaneous measurements of nine mucin genes. Specifically, a two-step procedure was designed that involved multiplex gene-specific PCR (nine mucin RT-PCR primer sets in addition to a housekeeping gene and other genes related to asthma and atopy) followed by real-time PCR on the generated cDNA product. The procedure was based on the PE Biosystems (Foster City, CA) ABI Prism 7700 sequence detection system, using a 96-well plate format. This approach was validated with 34 sets of genes, and no skewed representation of mRNAs was observed even after 25 cycles of RT-PCR amplification of cDNAs (data not shown). All the reagents including TaqMan universal PCR master mix and optical plates were purchased from PE Biosystems. The primers and probes for RT and TaqMan were designed with Primer Express software (PE Biosystmes), based on sequencing data from National Center for Biotechnology Information (NCBI, NIH) databases (Tables 2 and 3) and purchased from Biosearch Technologies (Novato, CA). Both sets of the primers were nested, and RT-PCR products were within a range of 250 bp. All PCR conditions were as recommended by the manufacturer, and the results are expressed as the number of mucin gene copies normalized to the number of the transferrin receptor gene copies (the housekeeping gene with constant expression in our system; in preliminary experiments the expression levels of transferrin receptor gene correlated well with other housekeeping genes, including glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). The copy number of each gene was derived using calibration curves, where C_t values (threshold cycle in real-time PCR that reflects mRNA copy numbers in a given RNA sample) were plotted against amplicon concentration in copy numbers (K. Livak, Sequence Detector User Bulletin 2, PE Biosystems) (26). Dilution curves confirmed the linear dependence of the C_t signal on the concentration of template RNAs, and revealed that the linear response of the TaqMan real-time PCR was most reliable when the C_t values were less than 38 (> 1 copy per reaction).

All measurements were made on coded samples that hid the identity of the subject. Data were entered into a computer spreadsheet program (Microsoft Excel; Microsoft, Redmond, WA) and exported to a statistics program (StatView; Abacus Concepts, Berkeley, CA) for descriptive and comparative statistics. The Mann–Whitney U test was used to compare data on goblet cell mucin stores in the airway epithelium and mucin concentrations in induced sputum. The unpaired t test was used to compare the copy numbers of genes for the nine mucin genes in the two populations. The Spearman rank order test was used to determine correlations between data. A probability value of < 0.05, using two-tailed tests, was considered significant.