Published ahead of print on February 16, 2006, doi:10.1165/rcmb.2005-0417OC
© 2006 American Thoracic Society DOI: 10.1165/rcmb.2005-0417OC Ciliary Beat Frequency Is Maintained at a Maximal Rate in the Small Airways of Mouse Lung SlicesDepartment of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts Correspondence and requests for reprints should be addressed to Dr. Michael J. Sanderson, Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue, North Worcester, MA 01655. E-mail: michael.sanderson{at}umassmed.edu
Ciliary beat frequency (CBF) is a key factor in the defense of the airways, and ATP can stimulate CBF by increasing intracellular calcium concentration ([Ca2+]i). However, the regulatory effects of ATP have been mainly studied in cultured or isolated epithelial cells from the large cartilaginous airways. The aim of this study was to evaluate the regulation of CBF in small airways of lung slices that are representative of in vivo tissue. Mice lungs were inflated with agarose and cut into thin slices with a vibratome. CBF in the small bronchioles was observed with differential interference contrast microscopy and quantified using high-speed digital imaging (at 240 images s–1). We found that the in situ organization of the ciliated cells was well preserved and that their CBF was high. We verified the fidelity of our recording system by analyzing rapid changes in CBF in response to temperature. However, we found that ATP had no effect on CBF, despite the fact that the [Ca2+]i, measured with confocal fluorescence imaging, was increased. Ionomycin and purinergic or  -adrenergic agonists also failed to increase CBF. Similar results were obtained in outgrowths of cells cultured from lung slices. By contrast, ATP increased the slower CBF of outgrowths of ciliated cells cultured from tracheal rings. Therefore, we conclude that CBF in intrapulmonary airways of mice is maintained at a maximum rate and cannot be further increased by agonist stimulation. These conditions would ensure that mucociliary clearance is constantly active to provide continuous airway protection.
Key Words: ATP • Ca2+ • high-speed digital microscopy • small airways
Mucociliary clearance is an important defense mechanism that clears foreign particles and chemicals from the airways to maintain healthy lungs. A key parameter determining the rate of mucus clearance is ciliary beat frequency (CBF). The density (cilia per cell and number of ciliated cells) and length of cilia are greater in the trachea than in small airways, and this correlates with an increase in mucus transport and CBF toward the oropharynx (1–5). This suggests that CBF is regulated throughout the airways to provide local control of mucociliary clearance. Unfortunately, this hypothesis is difficult to test in the smaller airways because of the inaccessibility of cilia. Consequently, most studies addressing the regulation of CBF have used cultured epithelial cells from the trachea or the large airways (cartilaginous bronchi) or trachea rings, and have found that CBF increases in response to a variety of stimuli, such as nucleotides (ATP, cAMP, UTP, cGMP) (6–12), mechanical deformation (13), NO (14, 15), and -agonists (16–18). Frequently, this increase in CBF has been associated with increases in intracellular calcium concentration ([Ca2+]i) (6, 9, 12–15, 19). By contrast, the signaling mechanisms that regulate CBF in the smaller airways remain unknown. In this article, we describe a unique method to study CBF in the smaller airways. Thin lung slices that retain the in vivo organization of the lung are prepared from mice and provide access to the cilia and epithelial cells at different positions of the bronchial tree. Differential interference contrast (DIC) microscopy and high-speed video recording could then be used to measure CBF. Because CBF is temperature dependent (20), we used this characteristic to validate our methodology for CBF measurements. With this approach, we determined that the CBF within the lung slices is normally maintained at a high rate of 20–25 Hz. The major result of this study is that CBF in the intrapulmonary airways of the lung slice cannot be further increased by extracellular ATP (1–500 µM), even though ATP increased the [Ca2+]i in the ciliated cells. By contrast, control studies confirmed that the slower CBF of cultured ciliated tracheal cells or tracheal rings could be increased by ATP. These results indicated that the cilia of intrapulmonary airways do not respond to increases in [Ca2+]i, a response confirmed with the Ca2+ ionophore, ionomycin. Similar results were obtained in rat lung slices. Collectively, these results suggest that the CBF of the intrapulmonary airways is not influenced by agents that increase [Ca2+]i. In view of the facts that the basal CBF of cultured cells is always slower than the CBF of lung slices, and that the maximal CBF rates induced in cultured cells approach that of intrapulmonary airway cilia, a simple explanation for the insensitivity of cilia to [Ca2+]i is that their beat frequency is maintained, in situ, at a maximal rate. With this design, mucociliary clearance in the lower airways would be constantly vigilant, and perhaps more reliable, for the immediate removal of microscopic foreign material.
Solutions and Chemicals Hanks' balanced salt solution (HBSS) without phenol red, FBS, and Dulbecco's modified Eagle's medium (DMEM) were obtained from GIBCO/Invitrogen Corp. (Carlsbad, CA). HBSS was supplemented with 25 mM HEPES (sHBSS; pH 7.4). Fluo-3AM and Fluo-4AM were obtained from Molecular Probes/Invitrogen Corp.. UTP, forskolin, ionomycin, hexokinase, adenosine deaminase, and pluronic F-127 were obtained from Calbiochem (La Jolla, CA). Sulfobromophthalein, ATP, isoproterenol, apyrase, agarose (type VII-A, low gelling temperature), and DMSO were obtained from Sigma-Aldrich Corp. (St Louis, MO). Fluo-3AM and Fluo-4AM were dissolved in DMSO and diluted in sHBSS to the final working concentrations. Final DMSO concentrations were < 0.1%.
Lung Slice and Tracheal Ring Preparation
Lung slices were prepared from BALB/C mice (7- and 9-wk-old; Charles River Breeding Labs, Needham, MA) killed by intraperitoneal injection of pentobarbital sodium (Nembutal), as approved by the institutional animal care and use committee of the University of Massachusetts Medical School. A solution of 6% agarose was dissolved in distilled water at 60°C, cooled to 37°C, and mixed with 2x concentrated sHBSS to give a 3% agarose–sHBSS solution at 37°C. The trachea was cannulated, and the lungs were inflated with warm agarose–sHBSS (
For consistency, the ciliary responses of similar-sized airways were initially compared. This comparison was achieved by collecting the first five lung slices that contained distal airways from different lungs lobes of each mouse. To compare the responses of the airway at different locations, a single lung lobe was sectioned into Lung slices were maintained in DMEM supplemented with 10% FBS, antibiotics, and antimycotics at 37°C in 10% CO2. FBS was added to the media to match the conditions of cultured cells. Experiments were also performed with slices maintained in medium without FBS, but no differences were observed.
Tracheal rings were also prepared from BALB/C mice. The trachea was removed, washed with sHBSS, and manually sliced into rings (
Cell Culture
Drug Application
Measurement of CBF with High-Speed Digital Microscopy
Measurement of Intracellular Ca2+ with Confocal Microscopy Lung slices or cultures were incubated with Fluo-3AM or Fluo-4AM (20 µM), 100 µM sulfobromophthalein (an inhibitor that prevents dye extrusion via anion exchangers), and 0.2% pluronic F-127 for 45 min at 30°C, followed by 45 min in sHBSS containing 100 µM sulfobromophthalein at 30°C. Loaded slices or cultures were mounted in a custom perfusion chamber, and imaging was performed using a video-rate confocal microscope (21, 25, 26). In brief, a 488-nm laser was used as the excitation wavelength, and the emitted fluorescence (> 510 nm) was detected by a photomultiplier tube and a frame-capture board to form an image. Fluorescence images were recorded to a hard disk using Video Savant software at 30 fps. Changes in fluorescence were analyzed by selecting an ROI of  5 x 5 pixels in a single epithelial cell. Changes in fluorescence (F) were normalized to the initial level of fluorescence (F0). Final fluorescence values were expressed as a ratio (F/F0). The maximum F/F0 ratio obtained was used to compare different experiments.
Statistical Analysis
Measurement of CBF in Small Airways of Mice To be consistent and allow the comparison of data from different airways in different lung slices, we primarily examined, unless otherwise noted, the responses of the distal small airways within peripheral lung slices. These airways had a diameter of 80 µm, and retained the morphologic and physiologic characteristics of in vivo airways (Figure 1). The airways were lined with a healthy cuboidal ciliated epithelium. Importantly, the airway lumen was devoid of agarose that might have interfered with CBF, but the periciliary layer was not preserved because the lumen was filled with sHBSS. The airways are surrounded by contractile SMCs and alveoli tissue. To measure CBF, the motion artifacts resulting from SMC contraction were reduced by stiffening the agarose used to stabilize the lung slice. This modification did not appear to affect the lung slice in any other way. The morphology of the lung slice did not substantially change during the period of use. Similarly, no substantial differences were observed in slices cut from different lobes of the lungs.
Lung slices were considerably thicker than cultured ciliated cells and, because the epithelium was viewed in profile, the movement of many overlapping cilia was observed simultaneously (Figure 1). Consequently, we established an imaging system using DIC optics to provide a relatively narrow depth-of-field, so that only a few cilia with good contrast were visible, and reevaluated and confirmed the accuracy of our CBF recording technique. The placement of the analysis points near the base of the cilia provided reliable measurements for CBF (Figures 1C and 1D). With a sampling rate of
CBF in Lung Slices Is Temperature Dependent
ATP Does Not Affect CBF Because many studies of cultured or trachea ciliated cells show that extracellular ATP enhances CBF (6, 9), we examined the effect of ATP on CBF in lung slices. Figures 3A–3C show examples of experiments performed at 30°C with 1, 5, and 10 µM ATP. Surprisingly, the CBF remained unaltered at any concentration of ATP tested (Figure 3D). By contrast, the SMCs of airways displayed some contraction, an indication that lung slices were sensitive to ATP. Repetitive stimulation with ATP (same or different concentrations) also did not change the CBF. Similar results were obtained at RT, but with a lower CBF. This lack of a response to ATP was not related to the age of the lung slices, because similar results were obtained with freshly isolated slices and slices maintained in medium for up to 3 d. It is also important to note that no differences in the basal CBF were observed between freshly prepared lung slices and slices maintained in medium for several days. We observed that CBF in rat lung slices was also not sensitive to ATP, a result suggesting that this response is not specific to mice (data not shown).
With serial slices, we also found no significant difference in the CBF when comparing the distal ( 80 µm in diameter: 22.73 ± 0.74 Hz, 10 cells, 3 mice), intermediate ( 150–210 µm in diameter: 22.46 ± 0.65 Hz, 10 cells, 3 mice), and proximal airways ( 270 µm in diameter: 21.26 ± 0.89 Hz, 10 cells, 3 mice), and that ATP (10 µM) did not change the CBF (distal airways: 23.03 ± 0.73 Hz; intermediate airways: 21.73 ± 0.93 Hz; proximal airways: 21.42 ± 1.16 Hz). Similar results were obtained with 100 µM ATP. These results indicate that the insensitivity of CBF to ATP was not dependent on the airway size or the location within the intrapulmonary tract.
Apyrase, Hexokinase, and Adenosine Deaminase Do Not Affect CBF
ATP Increases Intracellular Calcium in Airway Epithelial Cells
Ionomycin Increase of [Ca2+]i but Not CBF To determine if the lack of a CBF response, despite the increase of [Ca2+]i, was specific to ATP, we investigated the effect of a nonspecific increase in [Ca2+]i induced by the Ca2+ ionophore, ionomycin (8, 15). As expected, ionomycin induced large increases in fluorescence in the ciliated cells (Figures 5A–5C). This increase was homogenous throughout the cell, and was sustained for at least 1 min or more (Figure 5C). However, ionomycin had no effect on CBF: CBF was 19.17 ± 0.66 Hz before and 19.95 ± 0.79 Hz or 19.41 ± 0.63 Hz after treatment with 5 or 10 µM ionomycin (112 cells, 4 mice; Figure 5D). Similar to the effect of ATP, the SMCs displayed some contraction in response to ionomycin.
UTP, Forskolin, and Isoproterenol Did Not Increase CBF The lack of a response of CBF to ATP in the small airways suggests that the cilia were already at maximal CBF and could increased no further. To test this hypothesis, mouse lung slices were exposed to other purinergic agonists and -adrenergic agonists. UTP, forskolin, and isoproterenol (10–100 µM) all failed to increase CBF.
Extracellular ATP Increases CBF in Tracheal Rings
ATP Increases CBF in Cultures of Trachea Cells but Not in Cultures of Lung Slices A possible explanation for the differences in basal CBF and the responses in CBF and [Ca2+]i to ATP of airway epithelial cells is that the culturing process has altered the cell properties. To investigate this idea, tracheal rings and lung slices were plated onto collagen-coated coverslips and cultured for 5–10 d. After 5 d, outgrowths of ciliated cells from the tracheal rings and lung slices were observed. These cultured cells were maintained for a further 5 d, and no differences in CBF were observed with the age of the outgrowths of ciliated cells. In view of the idea that small airway cilia may be beating at a maximal frequency, it is important to note that the basal CBF of cultured cells in outgrowths from tracheal rings (13.23 ± 0.72 Hz; n = 56 cells, 6 mice) and lung slices (15.73 ± 0.70 Hz; n = 112 cells, 7 mice) was significantly lower in comparison with the CBF of uncultured tracheal rings (20.19 ± 0.84 Hz; n = 102 cells, 5 mice) and lung slices (21.65 ± 0.47 Hz; n = 112 cells, 5 mice; P < 0.05; Figure 6A). Similar to freshly prepared tracheal rings, the outgrowths of tracheal ciliated cells displayed a sustained increase in CBF in response to ATP (51 ± 5%, 5 µM ATP; Figure 6B). By contrast, but in keeping with the response of the uncultured lung slice, no change in CBF was observed in response to ATP in outgrowths of cultured cells from lung slices, even after 10 d of culture. However, ATP induced similar increases in [Ca2+]i in both cultured cells of tracheal rings and lung slices.
The observation that CBF can be increased in cultured or isolated ciliated cells from the large airways by a variety of stimuli has led to the hypothesis that mucociliary clearance is locally regulated throughout the airways. A major obstacle to the exploration of this hypothesis has been the incompatibility of the cilia of the smaller airways to microscopic analysis. To overcome this problem, we refined the preparation of mouse lung slices to gain access to the ciliated epithelium of the small airways. The lung slice preparation has several advantages, the most important of which is that it retains much of the in vivo organization of the lung (21). The activity of the ciliated cells can be studied without exposure to proteolytic enzymes, the disruption of cell contacts, or the influence of cell culture. In addition, it is relatively easy to prepare and maintain serial lung slices that allow the study of different parts of the airway. To measure ciliary activity, we adapted our previously proven technique of high-speed digital recording (9) by upgrading to DIC microscopy. However, to ensure that we accurately measured CBF in lung slices, we verified the fidelity of our technique by quantifying changes in CBF in response to temperature. With these calibration experiments, we confirmed the reliably of our method to measure rapid changes in CBF from one beat cycle to the next for extended periods (9). We emphasize this confidence in the quantification of CBF to underscore the fact that, in many experimental conditions, no changes in CBF occurred. CBF in isolated and cultured tracheal ciliated cells has been frequently found to be increased through increases in [Ca2+]i induced by ATP (6, 9, 11). By contrast, we consistently found, irrespective of concentration, that ATP had no effect on CBF in the intrapulmonary airways of mice. A prior stimulation of the ciliated cells within the airways by ATP or adenosine secreted into the airway surface liquid (27–29) might explain why CBF cannot be further increased in lung slices. However, apyrase, hexokinase, or adenosine deaminase, enzymes that metabolize ATP and adenosine, failed to slow CBF. In addition, in contrast to the air-filled lumen of the in vivo airway, the lumen of the airway of the lung slice is filled with fluid. This experimental condition, together with the constant perfusion of the preparation, would quickly dilute and remove any endogenous factors. Consequently, these results suggest that the CBF was inherently high and independent of secreted ATP or related compounds. However, a maximal increase in [Ca2+]i was induced by low concentrations of ATP. Although unexpected, the implication of these responses is that the cilia of small airways are insensitive to increases of [Ca2+]i. Support for this hypothesis is provided by the finding that, although ionomycin increased [Ca2+]i, it did not increase CBF. Similarly, ciliated cells in outgrowths cultured from the small airways of slices also did not show changes in CBF in response to ATP. The failure of increased [Ca2+]i to increase CBF also emphasizes that other endogenous factors, such as zinc, that act in conjunction with ATP to increase Ca2+ influx (30, 31), are unlikely to be responsible for the inherent high CBF. This insensitivity of CBF to regulation by ATP and [Ca2+]i was not specific to mice, because we found similar results with rat lung slices. Another study, by Hayashi and colleagues (32), performed with thicker rat lung slices (5–6 mm x 5–6 mm blocks) and lower CBF (9.2 ± 0.2 Hz), showed that ATP also failed to increase CBF in rat airways, whereas a significant increase could be observed in tracheal rings exposed to ATP. In contrast to our results, they found that ATP could not increase intracellular Ca2+ in rat lung slices. This insensitivity of CBF to ATP and [Ca2+]i appears to be a property of the ciliated cells lining the intrapulmonary airways of mice, because we found, in serial slices cut across the lung lobe, that CBF was also not stimulated in the distal or proximal airways. However, ATP increased CBF in both mouse tracheal rings and cultured ciliated cells from trachea rings. The important implication of these results is that the sensitivity of CBF to Ca2+ is related to the location of the cell in the respiratory tract, and that this characteristic is retained during culture. The possibility that this insensitivity to Ca2+ is related to the preparation of the lung slice is inconsistent with the numerous markers, including the morphological appearance, the Ca2+ response to ATP, and the ability of airways to contract and display dynamic Ca2+ signaling (21, 22), that indicate that the lung slice is representative of the in situ condition. From these results, it would appear that the ciliated cells acquire sensitivity to Ca2+ in the very large airways (cartilaginous bronchi or trachea).
A second important observation that implies an autonomous CBF is that the CBF in small airways was always high in comparison to the basal CBF of cultured cells in both this study and studies using different species (13 ± 0.3 Hz in rabbit tracheal epithelial cells [9]). In this study, we show that CBF of both the small airway and trachea cells is slowed down by being placed in culture. The reason for this slowed CBF is unknown, but it provides (at least in tracheal cells) the potential for subsequent increases in CBF (to approach the maximal rate of CBF) in response to agonists. Therefore, we propose that the insensitivity of cilia to ATP in lung slices is a result of CBF already operating at a maximum (at the optimal temperature). Our findings that other purinergic or Under conditions of maximal CBF, it would be predicted that inhaled particles, irrespective of size, would be quickly removed. This autonomous activity in the intrapulmonary airways of mice would seem to have an advantage over a local regulatory system that would have to be able to detect very small particles, such as bacteria, if a hazardous accumulation of material is to be avoided. Because of the convergence of the airway surface area, mucociliary loads increase toward the oropharynx. It is possible that, in the larger airways, local regulation of CBF, perhaps via mechanical stimulation (34), becomes more important and necessary to cope with the increased load. Although we found that rat intrapulmonary airways also displayed an autonomous CBF, the correlation of local regulation of CBF within the larger airways emphasizes caution when extrapolating these studies to larger animals, including humans. In summary, we demonstrate that a variety of agonists or increases in [Ca2+]i have no effect on CBF in small airways. We conclude that CBF is differentially regulated in the upper airways, and that culturing epithelial cells can modify CBF regulation. Finally, we propose that maximal CBF is maintained in vivo in small airways to sustain optimal mucociliary clearance.
This work was supported by National Institutes of Health grant HL70882 (M.J.S.). Originally Published in Press as DOI: 10.1165/rcmb.2005-0417OC on February 16, 2006 Conflict of Interest Statement: Neither of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form November 8, 2005 Accepted in final form February 3, 2006
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Abstract
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