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Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California
Submitted 28 July 2006 ; accepted in final form 19 September 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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innate defense; mucus
Based on serial reconstructions of human glands (19), CFTR immunohistochemistry, and direct observation with Nomarski differential interference contrast (DIC) microscopy, a four-compartment model has been developed for airway gland secretion (Fig. 1A) (13, 32). In this model, a gland comprises distal serous acini and mucous cell tubules, which all empty into a large diameter collecting duct that connects to the surface via an unbranched ciliated duct (19). According to a report based on a potent and well-characterized polyclonal antibody, CFTR is primarily located in serous acini (5); however, more recent work using sensitive monoclonal antibodies found abundant CFTR in the ciliated ducts but saw little or no CFTR signal in the serous acini (17).
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We next hypothesized that VIP/forskolin-stimulated secretion might actually originate from the CFTR-rich ciliated duct, just as pancreatic fluid secretion is known to be primarily of ductal origin (27); the studies reported here test that hypothesis. We used two different methods to determine if ciliated ducts either secrete (or absorb) significant amounts of fluid. We ligated the ducts at one or both ends and compared fluid transport across the ductal epithelium with that in the other compartments. We also directly monitored mucus flow rates within different compartments of the gland using time-lapse, DIC (Nomarski) microscopy, and particle tracking.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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290 mosM. Pieces of airway 2 cm2 were pinned mucosal-side-up, and the mucosa with underlying glands was dissected from the cartilage and pinned on a Sylgard-lined petri dish with the serosa faced up. Muscle fibers and connective tissues were dissected away from a set of submucosal glands, which were then either isolated with ducts intact (i.e., a small piece of airway surface was included with the duct orifice) or left in situ with ciliated duct still attached to the surface epithelium. Ciliated ducts are here defined as the unbranched portion of the duct that runs from the first branch point in the acinar rich area of the gland to the airway surface.
Gland isolation, duct ligation, and optical monitoring of duct diameters.
Submucosal glands were isolated so that the entire ciliated duct was accessible, and were then observed with a Wild stereoscopic microscope at x25 magnification with lateral illumination via a flexible, fiber-optic illuminator (model 190; Dolan-Jenner Industries, Lawrence, MA). Gland ducts were ligated with 22-µm nylon threads using either a single ligation near the orifice (Fig. 1B) or a second ligation between the ciliated and collecting duct. Ligated glands were pinned to a Sylgard-lined chamber of a glass slide, and gland duct diameters were monitored by digital imaging with a compound microscope using x2.5 or x4 objectives. The initial digital micrograph was made at 22°C and served as the control image against which all subsequent measures were compared. We tested responses to three conditions, each maintained for 20 min: 1) warming to 37°C in Krebs alone, 2) addition of 10 µM forskolin, or 3) with 10 µM carbachol. All measurements were made after 20 min, all were in the presence of 95% O2 and 5% CO2, and all but the first were at 37°C.
In situ gland ligation and optical monitoring of single gland secretion.
Glands were exposed as described above, but instead of isolating the glands they were left with ciliated duct still attached to the surface epithelium. Selected glands (up to 13 per tissue) were then ligated at the collecting/ciliated duct junction, leaving a larger number of adjacent, unligated glands to serve as controls. The tracheal mucosa was then prepared for optical monitoring as described previously (14). In brief, the tissue with attached glands was pinned mucosal-side-up in a Sylgard-lined plastic dish with the serosa in the bath (2 ml vol) and the mucosa in air. The tissue surface was cleaned, dried, and covered with a layer of water-saturated mineral oil; bubbles of mucus that formed at the orifices of the gland ducts in the oil layer were visualized by transillumination with an array of 10-mm diameter, ultra-bright, white-light-emitting diodes, which also revealed the glands and ligatures. Digital images were captured with a Nikon digital camera mounted on a Wild dissecting microscope. For stimulation, the tissue was warmed to 37°C at a rate of 1.5°C per minute while being continuously superfused with warmed, humidified 95% O2 and 5% CO2. Pharmacological agents were diluted to their final concentrations with warmed, gassed bath solution and added to the serosal side by complete bath replacement. The tissue was stimulated with 10 µM forskolin for 20 min followed by 10 µM carbachol unless indicated otherwise.
Monitoring fluid flow within specific compartments of single glands with DIC. Glands were isolated as described above and then further microdissected to prepare relatively clean but intact glands for optimal imaging. When judged suitable for imaging, the glands were transferred to microperfusion chambers on the stage of an upright Nikon Eclipse E600FN Series Microscope equipped with DIC and epifluorescence. They were continuously perfused with Krebs or Krebs-forskolin gassed with 95% O2 and 5% CO2 via a pressurized, eight-chamber, solenoid-actuated perfusion system (Automate, San Francisco, CA); temperature was maintained at 35–37°C using a TS-4 Peltier effect temperature controller that warmed the inflow tubing and chamber.
For digital imaging, the microscope is fitted with a Retiga-1300, cooled, 12-bit, color-Bayer Mosaic CCD camera with RGB Liquid Crystal Color Filter Module. The camera is interfaced with a high performance computer running Compix Image capture and analysis software.
For overview images of the gland, we used a x4 objective. For detailed studies of mucus flow, glands were imaged with a x40 water-immersion lens (numerical aperture 0.8, 2-mm working distance), and time-lapse digital imaging was used to monitor changes in mucus flow with frame rates of 1 frame/s to 2 frame/min. At this magnification, the field and focal plane are sufficiently restricted so that it is usually necessary to pick a single duct, tubule, or acinus for optical imaging.
Particle movements were first described during in situ studies of single airway glands by Ballard and colleagues (7). We assume as a first approximation that the net longitudinal movement of particles observed within the mucus can be used to measure the bulk flow of the mucus, and that, together with the duct or tubule diameter, the secretion rate can be calculated and expressed as picoliter per minute; if the measurement occurs in the final unbranched duct of the gland, it can be expressed as picoliter per minute per gland. We assume that particles will 1) accelerate if the surrounding epithelium is secreting, 2) decelerate if the surrounding epithelium is absorbing, and 3) be constant if the surrounding epithelium shows no net secretion or absorption.
Reagents. Compounds (Sigma) were made fresh or maintained at –20°C in the following solvents: carbachol and VIP in distilled water, indomethacin in ethanol, and forskolin in Me2SO. All were diluted 1:1,000 with bath solution (except indomethacin, which was diluted 1:10,000) immediately before use at the concentrations indicated.
Statistics. Data are means ± SE, and Student's t-test for unpaired data was used to compare the means of different treatment groups unless otherwise indicated. The difference between the two means was considered to be significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Both ciliated and collecting ducts showed larger increases after stimulation. As shown in Fig. 2C, the ciliated duct increased by 12.6% to 34.0 ± 3.6 µm when exposed to 10 µM forskolin and by 18.3% to 36.8 ± 5.4 µm after 10 µM carbachol. The corresponding increases in the collecting duct diameter were 13.3% to 48.0 ± 1.8 µm after forskolin and 26.2% to 50.8 ± 2.1 µm after carbachol. All changes were significantly different from the 37°C control (P < 0.05) except the ciliated duct response to forskolin (P = 0.08). We saw no visible mucus exiting near the ligation site, suggesting little or no leakage resulted from the ligations. Glands were swollen in all compartments but more prominently toward the distal (acinar) regions (see DISCUSSION). In sum, these experiments demonstrate that isolated, ligated glands continue to secrete fluid basally and in response to mediators, that more distal regions show a greater degree of expansion, and that isolated, ligated glands either do not leak or secrete at a rate that overwhelms any leakage.
Double ligations that isolate the ciliated duct demonstrate that secretion originates from distal regions of the gland. To determine if the fluid secreted by ligated glands originates wholly or in part from the ciliated duct, we made dual ligations at both the duct orifice and at the junction between the ciliated and collecting duct (Fig. 3A) in a series of glands that were then treated as before with temperature increase and forskolin and carbachol stimulation. Glands with larger ciliated ducts were chosen for these experiments. The average pig ciliated duct diameter under the control condition was 53.2 ± 3.4 µm; the collecting duct was 41.9 ± 11.8 µm. As shown in Fig. 3B, the doubly ligated (isolated) ciliated duct diameter showed minimal changes under all conditions, increasing by 0.5 ± 0.7% (0.2 ± 0.4 µm) when warming from 22 to 37°C, by 0.1 ± 1.9% (0.06 ± 0.93 µm) after 10 µM forskolin, and by 1.3 ± 1.5% (0.74 ± 0.79 µm) in 10 µM carbachol (n = 5, P > 0.35, not significant for all conditions). By contrast, the collecting duct expanded by 5.6 ± 5.6% (0.87 ± 1.6 µm) when warmed, by 16.5 ± 10.9% (4.2 ± 2.3 µm) in forskolin, and by 54.6 ± 8.8% (22.5 ± 6.5 µm) in carbachol (n = 5; P = 0.57, 0.11, and 0.018, respectively).
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Single ligations between the ciliated and collecting duct abolished fluid secretion from the open orifice of the ciliated duct. An alternative interpretation of the double ligation experiment is that ductal fluid secretion is stopped by negative feedback mechanisms that detect pressure in the duct. To address that issue, we left the duct orifice open but isolated the duct from the rest of the gland with a single ligation at the distal end of the duct where it originates from the collecting duct. Fluid secretion was then monitored in situ using the optical oil layer method (14). Mucus accumulation under oil at the orifices of ligated glands (isolated ducts) was then compared with neighboring, unligated glands in the same preparation (Fig. 4). We observed no secretion from the ductal openings of 23 ligated glands in experiments from three different pigs, even though the entire ciliated duct was intact in the ligated glands. By contrast, essentially all of the control, unligated glands showed mucous secretion.
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Origins of gland mucus secretion determined by DIC time-lapse imaging. We are pursuing a research program that uses DIC methods to study many aspects of airway gland secretion. As part of that program, we have taken advantage of particles that are observed in mucus (7) to track mucus flow through the various compartments of the airway submucosal glands. As stated in METHODS, particles flowing through a tube that is adding fluid either across the epithelium or via inlet tubes will accelerate, whereas particles in a tube that is doing neither will display a constant rate of flow. Here, we have concentrated on a comparison of particle flow in the acini and in the ciliated duct in response to forskolin stimulation; these are the two gland regions where immunocytochemistry has localized CFTR.
Particles originate in acini and show accelerated movement from the acini. Microdissected but otherwise intact human airway glands from donor trachea were used for these experiments. For all results reported here, the glands had been slowly equilibrated to 37°C and were then stimulated with 5 µM forskolin and allowed to reach steady-state secretion. By visual inspection at low power, particle movement was observed in all regions of the gland, and in all cases the particles moved from the acini toward the ducts (data not shown). When glands were injured, either inadvertently or deliberately, the flow of mucus was altered and could sometimes be seen exiting from the injured region. As will be shown below, this provided an additional method for localizing the origins of secretion. At higher power, particles could be tracked within individual acini (Fig. 5A). They always moved toward the proximal portion of the gland, and the calculated secretion rate at their position increased as they moved along the acini (Fig. 5, A and C). Particles were seen in 14 of 30 acini from 20 human subjects under all experimental conditions, always with the same general trends of distal-to-proximal movement and acceleration, but we observed wide variation in actual particle speeds. Only two acini in steady-state forskolin had particles that gave meaningful data (the particles stayed in focus and were recorded long enough to allow analysis of secretion rates over time).
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140 pl/min per gland (see METHODS) with virtually no change in secretion rate as a function of distance in micrometers along the duct (0.33 ± 0.25 pl·min–1·µm–1, n = 7 subjects; Fig. 7, A and B), consistent with that region of the gland neither adding nor subtracting volume from the flowing mucus. Particles were analyzed in ducts from seven humans, always with the same general trends of distal-to-proximal movement and constant speed, but we again observed wide variation in actual speeds of movement among different gland ducts.
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Nicked duct experiment shows static mucus between the nick and the duct orifice. Also in a single experiment, a small hole was made approximately halfway along a ciliated duct in an isolated pig gland, and mucus particles were observed at the region of the nick and again at the ductal orifice. As diagrammed in Fig. 8, particles flowed from the distal regions of the gland to the nicked region and then exited through the nick. By contrast, mucus in the duct between the nick and the ductal orifice was static, even though cilia were beating; this condition is not consistent with significant fluid secretion from the distal, ciliated portion of the duct.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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We used duct ligation and direct observation of particle movements to determine if the ducts contribute fluid to the gland mucus. Neither method provided any evidence for ductal secretion in response to forskolin, but instead indicated a more distal source for secretion. Direct observation of acinar particle movements showed that fluid secretion in response to VIP/forskolin originates in the serous acini with no evidence for fluid addition from the duct. Our results are consistent with an ultrastructural study of rat submucosal glands, where the mean number of parallel fibrils in tight junctions between serous cells (3.6 ± 0.4) was significantly smaller than those between mucous tubules (5.1 ± 0.6) or ciliated duct cells (8.5 ± 0.7). Furthermore, colloidal lanthanum permeated the tight junctions between serous cells up to the level of the acinar lumen, but did not penetrate between the mucous or ciliated cell junctions. These results suggest that tight junctions between serous cells are more permeable to small water-soluble solutes, which is consistent with a secretory function (25).
Some details relevant to gland function and the methods used. Glands ligated at the duct orifice showed larger volume expansion in the collecting duct than in the unbranched, ciliated duct. That could indicate that this region is more compliant. It is also possible that the expansion occurs because mucus forms a thixotropic gel (i.e., it develops gel strength when not sheared and liquefies when sheared). Thus, when static, such as at the end of a ligated duct, mucus gel strength should increase and resist the addition of further mucus. A similar explanation might have influenced the results of the nicked duct experiment (Fig. 8), where static mucus near the natural duct orifice was not moving even though the cilia were beating. However, even if filled with gelled mucus, the duct in this region should have expanded if fluid were being secreted by the ciliated cells, but it did not.
The ciliated portion of gland ducts contains ENaC subunits, raising the possibility that the duct may be absorptive and that increased absorption in CF ducts might contribute to CF gland dysfunction. In the present studies, we saw no evidence for volume absorption by the ducts, but we did not explicitly set out to test that hypothesis. Because of variability in particle speeds within the mucus (see below), we cannot completely eliminate the possibility of some volume absorption by the duct. However, that possibility has been addressed specifically by Joo et al. (10), who used ENaC blockers in an attempt to alter secretion rates from CF and normal glands. They were unable to rescue lost secretion from CF glands or to increase secretion from normal glands, and concluded that ENaC-mediated absorption does not occur in the ducts, possibly because ENaC is kept inactive by abundant Kunitz-type inhibitors present in gland secretions (10).
Several details from the particle-tracking observations raise points worth pursuing. Whereas tracking was adequate for the task of establishing an acinar origin for VIP/forskolin-mediated fluid secretion, we observed large variations in the size of particles and also noticed considerable heterogeneity in flow rates within the mucus: we observed "channels" of higher and lower flow rates that point to a considerable degree of structural complexity in the mucus as it is formed within the glands. It will be necessary to understand the origin and composition of the mucus particles and the different flow rates with the mucus stream if we are to fully appreciate the properties of normal and CF mucus.
The different patterns of CFTR expression revealed by different antibodies (5, 17) remain unresolved. The two sets of antibodies are directed at epitopes in different parts of CFTR that might be differentially exposed in different regions of the glands (W. Skatch, personal communication). It is possible that CFTR expression is indeed higher in ducts than in acini and that the monoclonal antibodies, which detected acinar CFTR in only about one-third of the samples (17), are simply inadequate to detect lower levels of acinar CFTR, which are nevertheless sufficient to mediate fluid secretion. A similar situation occurs in sweat glands, where very high levels of CFTR are present in the sweat duct alongside ENaC (3), where they jointly mediate salt absorption (20, 21). A lower level of CFTR is present in the sweat gland acini, where it mediates cAMP-stimulated fluid secretion that is lost is CF (2, 24). Indeed, we hypothesize that high densities of Na+ and Cl– channels are required to allow epithelia to absorb salt in excess of water, since the water permeability of membranes is relatively high even in the absence of aquaporins. The salt content of airway secretions has been a contentious issue (31), but in the only direct measures of ions in uncontaminated gland secretions, levels of Na+ and Cl– were significantly lower than bath values (9). Reduced levels of Na+ and Cl– were also found with bulk collections of nasal or tracheobronchial mucus after maneuvers that stimulated gland secretion (16).
We focused on just two immunolocalization studies with divergent results, but at least two other immunolocalization studies of CFTR in human airways have been carried out, and these also support an acinar location for CFTR. In a study of normal human nasal turbinates using MAb 24–1, a mouse IgG2a antibody directed against the four C-terminal amino acids of CFTR, Wioland et al. (33) observed intense staining of the apical membranes of serous cells, no staining of gland mucous cells, and weaker staining of surface epithelium. Jacquot et al. (8) applied anti-CFTR antibodies to human tracheal glands and saw immunolabeling along both the apical and basolateral plasma membranes of mucous cells and the secretory granules of serous cells. Using immunogold electron microscopy, the immunolabeling was more specifically associated with the membranes of serous cell secretory granules. A consistent result across these two studies is that staining with anti-CFTR antibodies was observed in gland serous cells.
In conclusion, forskolin stimulated acinar but not ductal fluid secretion. Because CF glands do not secrete in response to VIP/forskolin (11), we conclude that significant acinar fluid secretion is missing in such glands and is therefore by definition CFTR dependent. We also clearly observed acinar fluid secretion in response to carbachol and failed to observe ductal fluid secretion to carbachol. However, because carbachol induces significant fluid secretion in CF glands (9–12, 23, 26, 29, 32), the present studies do not allow us to make any statements about a possible role for CFTR in such secretion beyond showing that carbachol-stimulated fluid secretion does not have a ductal origin. Our results cannot eliminate a very minor component of ductal modification of fluid volume and do not address the possibility of ductal modification of fluid composition.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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