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* Cystic Fibrosis/Pulmonary Research and Treatment Center and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In airway defense, mucus clearance is supplemented by airway macrophages that patrol airway surfaces and eradicate inhaled bacteria in concert with antimicrobial proteins (6) and other proteins such as IgA and collectins (7). When these surface defense processes cannot contain an inhaled bacterial load, neutrophils are recruited from the blood to kill bacteria. As compared with alveolar pneumonias, neutrophils in airway infections have to penetrate mucus and move in three dimensions to capture and kill bacteria. However, there have been no investigations of neutrophil function in the three-dimensional environment that characterizes mucus-covered airway surfaces. Instead, studies of neutrophil migration in the airways have focused on how neutrophils migrate from the capillaries through the extracellular matrix and onto the epithelial surface, not how they move through the actual infection site, i.e., mucus (8, 9).
Accordingly, we designed experimental systems to investigate how neutrophils penetrate mucus to capture bacteria and whether these processes are affected by the abnormally thickened (concentrated) mucus that characterizes the CF airway. First, we examined two basic features of neutrophil motility in mucus of varying concentrations and containing a chemoattractant, i.e., chemotaxis and chemokinesis. The conventional devices to measure neutrophil migration in gels have measured migration in either the downward (with gravity) or lateral directions (10, 11, 12, 13). However, because 50% of airway surfaces face the "upward direction" (antigravitational) in vivo, we designed assays to measure both upward and downward neutrophil migration through mucus. These in vitro studies were complemented by in vivo studies in xenografts. Second, we measured the capacity of neutrophils to capture and kill bacteria in normal and thickened (concentrated) mucus using heat-killed and live bacteria. Finally, to mimic the in vivo situation, studies of neutrophil-bacteria interactions over a range of mucus concentrations were performed with both ciliated and nonciliated human airway epithelial cultures. For all studies, mucus was obtained from human airway epithelial cultures and processed to generate mucus concentrations that spanned normal and CF values.
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Materials and Methods |
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HBE cells were isolated and cultured under air-liquid conditions as previously described, with certain modifications (14). The modifications required to collect mucus included plating 1 x 106 passage-2 HBE cells on collagen-coated permeable supports (Transwell-Col, 24 mm in diameter, 0.4-µm pore; Costar). To prepare ciliated and nonciliated cultures for neutrophil migration studies, passage-2 HBE cells were seeded on Transwell-Col (12 mm). Day 5–14 cultures were nonciliated, and day 21–60 cultures were ciliated, as defined by the presence or absence of beating cilia with microscopy (TE300; Nikon).
Preparation of concentrated mucus
After HBE cells started to produce mucus, 200 µl of PBS were added to the apical surface, and 24 h later, all liquid (mucus) was aspirated. The material harvested from cultures was pooled and stored at 4°C. The mucus samples were placed in dialysis tubes (MWCO = 3500; Spectrum Laboratories) and concentrated with a polymer absorbent (Spectra/Gel; Spectrum Laboratories) for 1–5 days at 4°C until the mucus reached the target volume, which was calculated from the percent dry weight and the volume of the initial mucus sample. To generate an isotonic sample, the concentrated mucus was dialyzed against PBS (2 liters x 2) containing 500 µM MgCl2 and 800 µM CaCl2 at 4°C, then 1/100 volume of D-glucose solution (100 mg/ml) were added (final concentration, 1 mg/ml). The percent dry weight was calculated by measuring wet and dry weights with a microbalance (model 27; Cahn Electronic Balance) before and after drying the sample at 80°C overnight (15). Because of the difficulty in depleting harvested mucus samples of exact volumes, a 10% difference from the target concentration was allowed.
Determination of mucin content of mucus
The concentrations of mucins in HBE cell culture secretions were assessed by gel chromatography and differential refractometry (dn/dc). A value for dn/dc of 0.165 ml/g was used for mucins, which we have measured previously at 650 nm. To measure mucin concentrations, samples were diluted in running buffer in the range 1/20 to 1/40 to maintain a response within the dynamic range of the instrument, and a 500-µl sample was chromatographed on a column of bed volume (12 ml) containing Sepharose CL-2B (Amersham Biosciences). The column was eluted with 200 mM sodium chloride/10 mM EDTA at a flow rate 0.3 ml/min. The column effluent was passed through an in-line Dawn enhanced optimal system laser photometer coupled to a Wyatt/Optilab digital signal processing inferometric refractometer to measure light scattering and sample concentration, respectively. Light scattering and refractive index measurements were made continuously, with the light scattering being used only to identify and define the mucin peak. The captured data were integrated and analyzed with the Astra software provided with the Dawn. Optimum dilutions were sought by trial and error and then paired values were obtained to check reproducibility, which was typically within 5%.
Characterization of the structure and function of mucus samples
The topography of mucus samples of different concentrations was visualized with wheat germ agglutinin (lectin from Triticum vulgaris) conjugated to tetramethylrhodamine (WGA-Rh; Molecular Probes). A 100-µl sample of each mucus concentration was placed on a coverslip chamber (CoverWell 0.5 x 20 mm; Grace Biolaboratories) with 1 µl of 5 mg/ml WGA-Rh and visualized by confocal microscopy (Zeiss Laser Scanning Microscope 510; Carl Zeiss) with a x40 objective (numerical aperture, 1.3; oil immersion). Pore diameters in the mucus meshwork were estimated with a Metamorph image analysis system (Universal Imaging) by tracing the mucin fibers surrounding the pores (100 pores/image from three images per concentration) and calculating cross-sectional areas. Equivalent pore diameters were calculated as the diameters of perfect circles having the same areas.
Mucus transport was measured for the different percentage of solids samples on well-differentiated, fully ciliated epithelial cultures. As reported previously (2, 14), mucus is transported in a rotational fashion on the surfaces of cultures when mucus is physiologically hydrated, whereas concentrated mucus is stationary. Each mucus sample (30 µl) was mixed with 1 µl of a 0.2% suspension of fluorescent microspheres (yellow-green, carboxylate-modified, 1 µm in diameter; Molecular Probes) and placed on well-differentiated HBE cultures. Pictures were taken with an epifluorescent microscope (Eclipse TE300; Nikon) equipped with a digital camera (DVC1312M; DVC) with a x4 objective and a 5-s exposure.
Neutrophil chemotaxis in mucus
Neutrophils were isolated from normal healthy adult volunteers, as reported previously (16) (protocols approved by the University of North Carolina Institutional Review Board). Neutrophils, suspended in HBSS containing 500 µM MgCl2 and 800 µM CaCl2, were labeled with calcein-AM (10 µM) for 30 min at 37°C.
For the gravitational ("downward") chemotaxis measurements (see Fig. 2Ai), 10 µl of 100 nM fMLP (Sigma-Aldrich) were added to the bottom of 24-well culture plates and allowed to dry. Then, 200 µl of mucus (1.5, 2.5, or 6.5% dry weight) were added, and a Transwell (6.5 mm in diameter, 3.0-µm pore, polycarbonate; Costar) placed atop the mucus (Fig. 2Ai). A neutrophil suspension in mucus was prepared by adding 1.5 x 103 calcein-labeled neutrophils to 30 µl of mucus and added to the Transwell lumen. After 3-h incubation, the number of neutrophils that had migrated through the Transwell membrane into the lower compartment was quantitated with a fluorescent microscope (Eclipse TE300; Nikon).
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Neutrophil chemokinesis
After migrating to the airway surface from the submucosa, neutrophils contact the mucus covering the epithelial surface. Neutrophils can either migrate into mucus or move along the epithelial surface. Lateral motility (epithelial surface movement) of neutrophils was measured on nonciliated airway epithelial cultures by depositing a calcein-labeled neutrophil suspension (2 x 104 cells in 200 µl) on nonciliated airway epithelial culture surfaces for 30 min to allow neutrophils to sediment. The supernatant was gently aspirated, mucus samples (1.5, 2.5, and 6.5%) containing 10 nM fMLP added to the epithelial surfaces, and the preparations placed in a heated chamber (
TC3; Bioptechs) on an epifluorescent microscope. Movement of neutrophils was recorded at 1-min intervals for 15 min at 37°C, and the tracks analyzed with a Metamorph image analysis system (Universal Imaging). Four series of images with >10 neutrophils each were obtained and analyzed for each mucus concentration.
Neutrophil migration into normal and CF xenografts
Tracheal xenografts were created as previously described (17), with the following modifications. Briefly, rat tracheas were denuded of their own epithelium and both ends cannulated with polyethylene tubing. Between 0.5 and 1 million passage 1 or 2 human CF or non-CF airway epithelial cells were placed into the lumen, cannulas ligated with vascular clips, grafts implanted s.c. into the interscapular space of Nu/Nu mice, and allowed to mature for 14–19 days. The mice were then anesthetized, graft ends revealed surgically, ligatures removed, and the lumen injected with varying concentrations of mucus containing 100 nM fMLP. Xenograft model tracheas seeded with non-CF epithelia were injected with 2.5% mucus as a normal airway model, whereas tracheas seeded with CF epithelia were injected with 8% mucus as a CF airway model. After 6 h, grafts were removed and rapidly frozen for sectioning. Neutrophil number was counted with image analysis software after acquiring digital images of five sections for each xenograft. Xenograft sections with unepithelialized surfaces or inhomogeneous mucus distribution were excluded from analysis. The number of neutrophils was counted in the entire lumen and the outer half to calculate the peripheral to central ratio of neutrophil distribution. All procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee.
Preparation of bacteria
Heat-killed, fluorescently labeled Escherichia coli (K-12 strain, tetramethylrhodamine conjugate; Molecular Probes), preopsonized for 30 min with rabbit polyclonal Abs (Molecular Probes), was used to examine the capability of neutrophils to capture immotile (dead) bacteria. Pseudomonas aeruginosa was used to examine neutrophils for their ability to capture motile "live" bacteria. An overnight culture of P. aeruginosa PAO1 (18), grown on trypticase soy agar (Oxoid), was prepared for each assay. In addition, we also used a PAO1 strain carrying plasmid pTdK-gfp (19) (GFP-PAO1). This strain constitutively expresses the stable form of GFP. To maintain the plasmid, cultures were grown for each assay overnight on trypticase soy agar in the presence of carbenicillin (200 µg/ml).
Neutrophil capture of heat-killed bacteria in mucus on ciliated and nonciliated HBE cultures
To test whether neutrophil motility (chemotaxis and chemokinesis) in mucus of varying concentrations affected bacterial capture, we incubated calcein-labeled neutrophils with preopsonized, heat-killed E. coli (tetramethylrhodamine-labeled). Two hundred microliters of a neutrophil suspension (2 x 104 cells) were added to the apical surface of nonciliated HBE cultures. After the neutrophils had sedimented onto the surface, the supernatant was gently aspirated, and bacteria suspended in mucus (2 x 105 bacteria/50 µl of mucus) were placed on the epithelial surface. After overnight incubation, pictures were taken with epifluorescence and confocal microscopes (x-z scanning). The number of neutrophil-associated bacteria was counted with an epifluorescent microscope. For x-z confocal scanning, epithelial cells were counterstained with 5 µM calcein-AM.
Because preliminary experiments revealed that beating cilia prevented neutrophil sedimentation onto the surface of ciliated cultures, neutrophils (2 x 104) and bacteria (2 x 105) were mixed and suspended in 50 µl of mucus and placed on ciliated cell cultures. After overnight incubation, the number of neutrophil-associated bacteria was counted with an epifluorescent microscope. The percent dry weight of the mucus was not changed significantly during the incubation (data not shown).
Neutrophil capture of live P. aeruginosa in mucus
We used GFP-PAO1 to measure capture of live bacteria by neutrophils in mucus. Two hundred microliters of a neutrophil suspension (5 x 105 cells/ml) were placed in a plastic coverslip chamber (0.5 x 20 mm; Grace Biolaboratories). After neutrophils had sedimented onto the plastic surface (
30 min), the supernatant was aspirated, and 100 µl of mucus containing 1 x x06 GFP-PAO1 (bacteria:neutrophils = 10:1) were added to the chamber. After a 2-h incubation at 37°C, the number of neutrophil-associated bacteria was counted with epifluorescence microscopy. For this experiment only, neutrophils were labeled with a red fluorophor (5-(and-6)-chloromethyl SNARF-1; Molecular Probes).
Neutrophil killing of P. aeruginosa in mucus on ciliated and nonciliated airway cultures
For all experiments with live bacteria, cell cultures were washed with PBS 24 h before the experiment and incubated with cell culture medium in the antibiotic-free basolateral bath. For nonciliated cell cultures, 200 µl of a neutrophil suspension (1 x 105/ml) were added to the apical culture surface. After neutrophils sedimented onto the surface, the supernatant was aspirated, and bacteria suspended in mucus (500 bacteria/50 µl of mucus) were added. The ratio of neutrophils to bacteria was 40:1. In preliminary experiments, ratios > 10:1 exhibited significant bacterial killing. After a 2-h incubation at 37°C, the number of live bacteria was determined by serial dilution and plating on sheep blood agar plates (BD Biosciences). The ratio of bacterial number 2 h after plating, with vs without neutrophils, was used to express bacterial killing mediated by neutrophils.
When ciliated cultures were used, neutrophils (2 x 104), bacteria (500), and mucus (50 µl) were mixed before addition to the cell cultures. The sample was then placed on ciliated bronchial epithelial cultures. Control experiments were performed with a mucus-bacterial mixture without the addition of neutrophils. After a 2-h incubation at 37°C, the mucus was harvested with PBS containing 10 mM DTT (20), the number of live bacteria determined by serial dilution and plating on 5% blood agar plates, and the percent bacterial number used to measure neutrophil effect, as above.
Characterization of mucin species by HPLC ion-exchange chromatography
Mucus samples were dialyzed into urea reduction buffer (6 M urea containing 0.1 M Tris/5 mM EDTA (pH 8.0)) and treated with 10 mM DTT for 2 h at 37°C. Iodoacetamide was added to a final concentration of 25 mM, and the mixture was left in the dark for 30 min at room temperature. The reduced samples were chromatographed on an Amersham Biosciences MonoQ HR 5/5 column, using Amersham Biosciences, Ettan LC high-pressure chromatography system. The column was eluted with a linear gradient of 0–0.75 M lithium perchlorate/10 mM piperazine in 6 M urea (pH 5.0) with a flow rate 1 ml/min, and 0.5-ml fractions were collected. Aliquots from the fractions were transferred to the nitrocellulose membrane by slot blotting using a Schleicher and Schuell blotter. Mucins were detected on the membrane by standard chemiluminescence assay, using the polyclonal antisera (MAN-5AC1, MAN-5B1) to MUC5AC and MUC5B mucins, respectively. The relative intensity of the slot blot bands was measured by densitometry and quantitated by ImageQuaNT (Molecular Dynamics) software.
Characterization of CF vs normal mucin species by western blotting after agarose gel electrophoresis
Mucins may be disperse in size and charge density due to differential glycosylation. Thus, electrophoretic behavior cannot be matched simply to m.w. and or size. However, combined with ion-exchange chromatography, it does yield a qualitative idea relating mucin size distribution to charge density, i.e., the homogeneity or heterogeneity of the preparation. This is how the technique was used here. Mucins fractionated by ion-exchange chromatography were electrophoresed in 1.0% (w/v) agarose gel (15 x 15 cm) in 40 mM Tris-acetate/1 mM EDTA (pH 8.0) containing 0.1% (w/v) SDS. Electrophoresis was performed in a horizontal gel apparatus using Bio-Rad SubCell GT (75 ml of gel, 1500 ml of buffer, 82 V, and 2.5 h at room temperature). After electrophoresis, subunits were transferred to nitrocellulose by vacuum blotting in 0.6 M sodium chloride/0.06 M sodium citrate, using an Amersham Biosciences VacuGene XL at a suction pressure of 45 mbar for 1 h before detection of mucins (21). Mucins were detected by using polyclonal antisera MAN-5AC1 and MAN-5B1 raised against MUC5AC and MUC5B mucins, respectively (22).
Neutrophil functions in CF airway mucus
Mucus from CF airway epithelial cultures was collected, concentrated, and dialyzed as stated above, except 300–500 µl of PBS were needed to collect mucus due to hyperabsorption by CF airway epithelia over the 24-h period. Three experiments, gravitational and antigravitational neutrophil chemotaxis and bacterial killing by neutrophils on ciliated surface, were repeated using CF mucus with varying concentrations (1.5, 2.5, and 6.5% solids) to test whether neutrophil functions are dependent on mucus concentration or on CF or non-CF origin.
Statistics
Unless otherwise stated, all data are expressed as the mean ± SE. Comparisons among mucus samples of three concentrations were made with ANOVA and Tukey test. Comparisons were made with Student’s t test between normal and CF-like mucus samples with p < 0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To determine the biochemical composition of mucus samples of varying concentrations, total mucins within mucus samples of varying concentrations were measured. In all the mucus samples, mucins constituted 60% of the nonionic components (Fig. 1A). As mucus was concentrated from 1.5 to 6.5% solids, mucin concentration increased >10 times from 2.64 to 31.2 mg/ml. Given the presence of 1% salt in isotonic mucus samples, the mucin concentration should theoretically increase 11 times over this concentration range.
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We also measured the transport of mucus on ciliated cultures as a functional characterization of these mucus preparations. Microspheres embedded in 1.5% mucus were transported in a complex fashion, with variations in bead vector and velocity (Fig. 1Ci). Fluorescent microspheres trapped in 2.5% mucus exhibited organized rotational movement, with a velocity similar to that reported in vivo (14) (Fig. 1Cii). In 6.5% mucus, microspheres were poorly transported (Fig. 1Ciii), consistent with the concept that 6.5% solids mucus is abnormally concentrated.
Neutrophil chemotaxis
Neutrophil chemotaxis in the direction of gravity was measured between two compartments separated by a 10-µm-thick polycarbonate membrane with 3.0-µm pores. The number of neutrophils migrating to the lower compartment was greatest in 1.5% mucus (28.3 ± 5.2 cells/well), smaller in 2.5% mucus (0.4 ± 0.2 cells/well), and absent in 6.5% mucus (0 ± 0 cells/well) (Fig. 2Aii) (ANOVA, p < 0.0001 and Tukey test, p < 0.01 for both 1.5 vs 2.5 and 1.5 vs 6.5%).
To test whether neutrophils could migrate against gravity in mucus, neutrophil chemotaxis was measured in a chamber with mucus separating neutrophils (bottom) from a fMLP source (Fig. 2Bi, top). In contrast with the experiments in the direction of gravity, only 2.5% mucus supported neutrophil movement against gravity (Fig. 2Bii) (ANOVA, p < 0.0001 and Tukey test, p < 0.01 for both 1.5 vs 2.5 and 2.5 vs 6.5%).
Neutrophil chemokinesis
To measure the lateral motility of neutrophils on nonciliated epithelial surfaces covered with mucus of varying concentrations, neutrophils were tracked at 1-min intervals for 15 min (Fig. 3A) and mean velocities calculated (Fig. 3B). Chemokinetic motility was significantly lower on surfaces covered by 6.5% mucus than 1.5 or 2.5% mucus (Fig. 3B). Because neutrophils could migrate in the z-axis in 2.5% mucus (Fig. 2Bii), the mean velocity of neutrophils tracked in the two-dimensional images was likely underestimated.
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Neutrophil motility was then examined in an in vivo airway model, i.e., the tracheal xenograft that incorporates neutrophil migration from the vascular compartment, through the epithelium, and into an airway lumen filled with mucus. Neutrophils migrated into tracheal xenografts (both normal and CF models) filled with mucus containing fMLP after a 6-h incubation. The neutrophil distribution was homogeneous in normal xenografts (periphery:centerratio 
1; Fig. 4, A and C). In contrast, neutrophil migration into mucus was slowed at the periphery of the lumen in CF xenografts (Fig. 4B), with significantly fewer neutrophils reaching to the center of the mucus plug (Fig. 4C). Thus, it appears that concentrated mucus presents a significant barrier to neutrophil migration in vivo.
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To examine whether the low motility of neutrophils in concentrated mucus also degraded their capacity to capture bacteria, we first studied neutrophil capture of heat-killed bacteria as a model of immotile bacteria in mucus of varying concentrations. Following overnight incubation with 1.5% mucus (Fig. 5, Ai and Bi), heat-killed bacteria sedimented from the mucus and both neutrophils, and bacteria were concentrated near the epithelial surface. In contrast, both neutrophils and bacteria were detected throughout the mucus layer when the epithelial surface was covered by 2.5% mucus (Fig. 5, Aii and Bii). However, in both mucus concentrations, the distribution of neutrophils overlapped that of bacteria. With 6.5% mucus, the neutrophils were predominately located on epithelial cell surfaces, whereas bacteria were distributed throughout the mucus layer (Fig. 5, Aiii and Biii), i.e., their distributions did not overlap.
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On ciliated epithelia (Fig. 5D), the results were similar to those with nonciliated epithelia. The number of bacteria captured by neutrophils was highest in 1.5% mucus (188.3 ± 48.6 bacteria/100 neutrophils), although this number was less than the number of bacterial captured on nonciliated surfaces. The reduced capture on ciliated cultures likely reflected the reduction of sedimentation of bacteria onto cell surfaces due to ciliary beating. The 2.5 and 6.5% values were higher relative to nonciliated cultures, probably due to premixing of neutrophils with bacteria. However, the number of bacteria captured in 2.5% mucus (60.7 ± 4.9 bacteria/100 neutrophils) and 6.5% mucus (21.3 ± 10.3 bacteria/100 neutrophils) was reduced compared with 1.5% mucus.
Capture of P. aeruginosa in mucus
We then examined neutrophil capture of live motile bacteria in mucus of varying concentrations, using a PAO1 strain of P. aeruginosa. Bacterial velocity was highest in 1.5% mucus and lowest in 6.5% mucus (data not shown). After 2-h incubation of neutrophils with GFP-PAO1, the number of bacteria associated with bacteria was counted. Neutrophils captured more GFP-PAO1 after a 2-h incubation in 1.5% mucus (120.3 ± 6.5 bacteria/100 neutrophils) than in 2.5% mucus (48.3 ± 3.3 bacteria/100 neutrophils) (Fig. 6A). In 6.5% mucus, the motility of both bacteria and neutrophils was significantly reduced, resulting in only four bacteria being captured by 100 neutrophils.
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Neutrophil killing of bacteria in mucus was studied in ciliated and nonciliated airway epithelial cultures, mimicking the in vivo conditions of airway bacterial infection. We used a P. aeruginosa PAO1 strain to measure bacterial killing activity by neutrophils in 1.5, 2.5, and 6.5% solids mucus, using 500 live bacteria to mimic early infection. On nonciliated epithelia, neutrophils killed bacteria sufficiently well to reduce bacterial number almost in half in 1.5% mucus (57.1 ± 2.1% survival) and in 2.5% mucus (52.6 ± 4.6% survival). In contrast, neutrophils were completely ineffective in reducing bacterial numbers in 6.5% mucus (139.4 ± 29.3% survival) (Fig. 6B). Similar results were obtained on ciliated epithelial cultures (Fig. 6C). The number of bacteria was reduced significantly by incubation with neutrophils in 1.5% mucus (55.7 ± 4.7% survival) and in 2.5% mucus (50.6 ± 3.5% survival) but not in 6.5% mucus (103.6 ± 21.6% survival) (Fig. 6C).
Comparison of biochemical properties of CF vs normal mucus
Biochemical properties of CF and normal mucus were measured to determine whether there were any differences that might affect neutrophil migration. CF and non-CF mucins were compared for the quantity and characteristics of surface charge. Neither ion-exchange chromatography (Fig. 7A) nor agarose gel electrophoresis (Fig. 7B) detected differences in charge density or mass in CF vs normal mucins that might affect neutrophil migration.
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Mucus from CF airway epithelial cultures was collected and concentrated to test whether there were any CF mucus-specific effects on neutrophil functions. Neutrophil chemotaxis in the gravitational and antigravitational directions was similar in CF to non-CF mucus (Fig. 7, C and D) (downward, ANOVA, p = 0.00019 and Tukey test, p < 0.01 for both 1.5 vs 2.5 and 1.5 vs 6.5%; upward, ANOVA, p = 0.0045 and Tukey test, p < 0.05 for 1.5 vs 2.5% and p < 0.01 for 2.5 vs 6.5%). Neutrophils migrated best in the gravitational direction in 1.5% solids mucus and not at all in 6.5% solids mucus, whereas mucus of 2.5% solids, but not 6.5% solids, supported neutrophil migration in the antigravitational direction. Bacterial killing by neutrophils was also repeated with CF mucus of varying concentrations on ciliated airway surfaces. Similar to the result with non-CF mucus, the number of bacteria was reduced by neutrophils over 2 h in 1.5 and 2.5% mucus but not in 6.5% mucus (Fig. 7E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The mucus layer covering airway surfaces is a non-Newtonian fluid with complex viscoelastic and structural properties. The structure and biophysical properties of mucus are imparted by the mucin macromolecules that normally comprise 0.2–2% of solids content of this layer (Fig. 1A) and are organized as meshes with definable porosities (Fig. 1B). Mucus viscoelastic properties and the mesh organization appear well adapted under normal circumstances to trap and remove inhaled particles and bacteria from the lung. In CF, the abnormally concentrated mucus is predicted to have complex effects on viscoelastic properties and reduce the equivalent mesh size.
The selection of "normal" mucus, as indexed by concentration (percentage of solids) and CF-like concentrated mucus, is not straightforward because the shallow depth of ASL (<50 µm) makes sampling difficult. However, we estimated the percent dry weight (percentage of solids) of normal mucus at 1.5–2.5% and of thick CF-like mucus at 6–15%, based on data reported from cell culture (26), a mouse model (4), and human studies (27, 28, 29, 30, 31, 32, 33). Mucus of 1.5% solids may be less than the normal optimal concentration because the network of mucin fibers and transport were heterogeneous (Fig. 1, B and C). However, this concentration could mimic the situation in small airways where there are few mucin-producing cells (34, 35) and little influx of mucus from more distal airways. The 2.5% mucus, which exhibited normal transport (Fig. 1C), may reflect the concentration of mucins that occurs in larger airways due to increased mucin secretion and ASL volume absorption (36). The higher concentration of mucus we operationally chose to mimic CF, 6.5%, likely represents the minimum concentration found in CF airways before infection (29, 30, 32, 33). Mucus transport assays demonstrated that 6.5% mucus was too thick to be transported by cilia, mimicking a key feature of CF mucus (2).
We estimated the size distribution of the mesh pores from the images acquired with confocal microscopy (Fig. 1B). There are limitations of this method. For example, the images obtained were thin (0.5-µm depth) sections of a three-dimensional lattice. Therefore, all mucin fibers defining pore openings might not appear in a single image, which would overestimate pore size. In contrast, fibers seemingly defining pores may not actually interact with one another because of the poor z-axis resolution with confocal microscopy, which would underestimate pore size. However, the pore size measurements of mucus of different concentrations are consistent with our functional data. Most pore diameters of 1.5% mucus (>10 µm) and some of 2.5% mucus (1.6–14.1 µm) were large enough for neutrophil (10–15 µm in diameter) penetration, whereas pores of 6.5% mucus were much smaller (<0.2 µm), i.e., smaller than a neutrophil or a bacterium.
Neutrophil motility in the presence of a chemoattractant varied dramatically as a function of mucus concentration and mesh pore size in vitro and in vivo. In in vivo xenograft models of mucus plugs, neutrophil migration was inhibited by thickened airway mucus, forcing neutrophils to stay near epithelial surfaces (Fig. 4). This result is compatible with the data from three-dimensional in vitro motility analysis.
In the modified chemotaxis chambers (Fig. 2), 1.5% mucus proved to be the optimal concentration for neutrophil migration in the direction of gravity, perhaps reflecting a minimum inhibition of neutrophil motility due to the large mesh pores relative to the size of neutrophils (Fig. 1B) and/or low mucus viscoelasticity. Interestingly, only a small number of neutrophils migrated with gravity through 2.5% mucus as compared with 1.5% mucus. We speculate that as the concentration of mucus increased from 1.5 to 2.5%, the density of mucin strands increased sufficiently to produce a population of pores sufficiently small to inhibit neutrophil entry into Transwell membrane pores. The absence of detectable motility in 6.5% mucus was likely caused by a mucus mesh pore size too small to allow neutrophil penetration.
In contrast, neutrophil migration in the antigravity direction was markedly faster in 2.5% mucus than in 1.5 or 6.5% mucus (Fig. 2B). We speculate that 1.5% mucus did not support migration because the mucus was too dispersed to provide anchoring supports (pore size > 10 µm) for upward motility, as reported for studies of three-dimensional neutrophil motility in collagen gels (10, 11). Mucus of 2.5% solids was filled with pores that were in the range of porous membranes of conventional chemotactic chambers (1–8 µm in diameter). We speculate that this pore size was sufficiently large to allow neutrophil penetration but small enough to prevent neutrophil slippage induced by gravity. We did not identify the mode of interaction of neutrophils with 2.5% mucus in this study, i.e., whether neutrophils dragged the mucin network, widened the pores of the network, or digested the mucin protein, as in collagen gels (37), or simply changed their shape to penetrate the mesh pores. The absence of neutrophil motility in the antigravity direction in 6.5% mucus, again, likely reflected an environment with a mesh pore size too small for neutrophils to enter (Fig. 1B).
Neutrophil chemokinesis on epithelial cell surfaces likely plays a significant role in capturing bacteria that escape the mucus blanket and sediment onto airway epithelial surfaces, especially in deciliated or goblet cell-metaplastic areas less well defended by mucus clearance. Neutrophils moved efficiently in search for bacteria on airway epithelia covered by mucus of 1.5% solids (Fig. 3B), reflecting little inhibition of lateral motility of neutrophils due to large mesh pores (Fig. 1Bi). Thick mucus (6.5% solids), with no detectable pores (Fig. 1Biii), inhibited lateral movement of neutrophils (Fig. 3B), preventing them from capturing bacteria sedimented or attached to airway epithelial surfaces. The relevance of this observation to in vivo situations was borne out in the xenograft experiments that showed concentration of neutrophils at the epithelial surfaces, which reflected inhibition of vertical movement of neutrophils, in 6.5 vs 2.5% mucus (Fig. 4).
In normal lung defense, inhaled bacteria are deposited and trapped in mucus. In diseases of failure to clear mucus, the chronic airways infection is localized within the mucus layer (5, 38). Despite this pathogenesis in the lung and similar infection of mucus by H. pylori in the stomach (39, 40, 41), there are a few studies of the role of neutrophil capture of immotile bacteria in a mucus environment. Therefore, we developed protocols to reproduce the in vivo environment in which neutrophil motility in three dimensions within mucus was required to capture heat-killed bacteria. In 2.5% mucus, neutrophils moved both upward and laterally to capture bacteria (Fig. 5Aii). In contrast, in 1.5% mucus, neutrophils moved primarily laterally, requiring bacteria to sediment onto the epithelial surface for capture (Fig. 5Ai). Despite the restriction to vertical movement (Fig. 2Bii), neutrophils captured more bacteria in 1.5 than 2.5% mucus (Fig. 5C), reflecting the combined effects of higher lateral motility rates (Fig. 3B) and greater bacterial sedimentation that concentrated bacteria and neutrophils in the same plane (Fig. 5, Ai and Bi). In 6.5% mucus, the limited neutrophil motility in any direction and the absence of sedimentation of bacteria accounted for the virtual absence of bacterial capture. Similar relationships in neutrophil capture of immotile (heat-killed) bacteria were observed in the mucus of different concentrations on ciliated surfaces (Fig. 5D).
Finally, we tested whether neutrophil efficacy of capturing and killing motile bacteria in mucus was degraded in the mucus of abnormal concentration. In all three experimental approaches, 6.5% (concentrated) mucus inhibited neutrophil-mediated bacterial killing (Fig. 6). These data, plus those on bacterial capture (Fig. 5), led us to speculate that CF-like thickened mucus promotes the early acquisition of bacteria. These findings do not predict an early selection for a single type of bacteria, and indeed, CF infants are first infected by multiple different organisms, including both immotile (Staphylococcus) and motile (H. influenza, Pseudomonas) organisms. Only later, in the complex milieu of the CF chronically infected airway, will other factors, including mucus concentration and O2 tension (5), allow P. aeruginosa to predominate (see below).
Our experiments performed in concentrated normal mucus were based on the hypothesis that concentration is the critical variable characterizing CF mucus (2, 4, 25). However, it is possible that other CF mucus-specific factors inhibited neutrophil functions as well. Differences between CF and non-CF mucus have been reported, including that CF mucins is more sulfated than non-CF mucins (42), which could affect fMLP diffusion and/or neutrophil migration. However, it is not clear whether these differences are derived from CF transmembrane conductance regulator-specific genetic defects or from chronic infection and inflammation (43).
Therefore, we compared mucin gene products and their charge secreted from normal and CF cultures. Ion-exchange chromatography and agarose electrophoresis revealed that no differences were detected in the negative charge of either mucin species (MUC5AC, MUC5B) secreted by CF and normal airway epithelia (Fig. 7, A and B). Thus, we could find no biochemical evidence that normal and CF mucins differed in the absence of infection.
To directly test neutrophil functions in CF-derived mucus, we next repeated three experiments using CF-derived mucus concentrated to 1.5, 2.5, and 6.5% solids (Fig. 7, C–E). Neutrophil chemotaxis and bacterial killing capacity in concentrated (6.5% solids) mucus and in 1.5 and 2.5% mucus from CF cultures mirrored precisely the functions of neutrophils in mucus obtained from normal cultures over the same range of mucus concentrations. Although we could not repeat all the experiments with CF mucus due to limited supply of CF bronchial cells, these results strongly indicate that mucus concentration, rather than source, inhibits neutrophil functions in airways.
The failure of neutrophils to kill bacteria in 6.5% mucus (normal or CF) may have implications not only for the early bacterial acquisition aspect of CF pathogenesis but also the persistent, excessive inflammatory response that characterizes later stages of the pathogenesis of CF airways disease. P. aeruginosa that proliferates in mucus causes inflammation by release of its own products and stimulation of host cellular cytokine production (44, 45). Components of each system recruit neutrophils from the vascular circulation to airway lumens. If the mucus is too thick for neutrophil penetration to kill bacteria and resolve infection, activated neutrophils trapped at the edges of thickened mucus masses could cause adverse effects. For example, neutrophil elastase, released during "frustrated phagocytosis," causes epithelial damage, Ig degradation, and mucus hypersecretion, all components of the uncontrolled inflammation that eventually leads to airway wall destruction (33, 46).
In conclusion, neutrophils migrate through normal respiratory mucus to search for and capture bacteria that have deposited in the mucus layer or have sedimented through this layer onto the airway epithelial surfaces. In contrast, the thick (concentrated) mucus in CF limits neutrophil motility and prevents bacterial capture and killing, likely contributing to both the early (acquisition) and chronic phases of airway bacterial infection. Thus, these studies strongly suggest that a key contribution of low ASL volume to the pathogenesis of CF airways disease is via the abnormally concentrated "thick" mucus layer characteristic of this disease that allows bacteria to evade the neutrophil component of lung defense.
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1 This work was supported by the National Institutes of Health and the Cystic Fibrosis Foundation. 
2 Address correspondence and reprint requests to Dr. Hirotoshi Matsui, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599-7248. E-mail address: hmatsui{at}med.unc.edu
3 Abbreviations used in this paper: CF, cystic fibrosis; ASL, airway surface liquid; HBE, human bronchial epithelial; WGA-Rh, wheat germ agglutinin conjugated to tetramethylrhodamine.
Received for publication January 30, 2004. Accepted for publication April 29, 2005.
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