In vitro Studies of a Distillate of Rectified Essential Oils on Sinonasal Components of Mucociliary Clearance

Solutions

Myrtol^® was supplied in pure form by the sponsor of the study, G. Pohl-Boskamp (Hohenlockstedt, Germany). The stock solution was diluted 1:1 with 100% ethanol to solubilize the product and then appropriate dilutions were attained in Hank's balanced salt solution. Initial dilutions were performed to 0.5 and 0.1% and physiological efficacy evaluated. Serial dilutions were performed until no effect was seen in comparison with the vehicle control.

Sinonasal ALI Cultures

Cultures were prepared as previously described.^8–10 Briefly, patients were recruited from the Division of Rhinology of the Department of Otorhinolaryngology–Head and Neck Surgery at the University of Pennsylvania and the Philadelphia Veterans Affairs Medical Center with full approval of both institutional review boards. Informed consent was obtained during the preoperative clinic visit or in the preoperative waiting room. Y. Lai and D. Dilidaer contributed equally to this work. Selection criteria for recruitment were patients undergoing sinonasal surgery. Exclusion criteria included a history of systemic diseases such as Wegener's, sarcoid, cystic fibrosis, immunodeficiencies, and use of antibiotics, oral corticosteroids, or antibiologics (e.g., omalizumab) within 1 month of surgery. Initial experiments to determine dilutions with physiological sequelae were performed in ALI cultures from non-CRS patients. Then, direct comparison of CRS versus non-CRS ALI cultures showed no difference in cilia stimulation; thus, ensuing experiments did not differentiate disease status of source material. Additionally, our prior work has indicated that cilia dynamics are not anatomically distinct ¹¹ and, thus, location within the sinonasal cavity of the source material was not maintained. Sinonasal mucosal specimens were acquired from residual clinical material obtained during sinonasal surgery and transported to the laboratory in saline placed on ice. ALI cultures were established from human sinonasal epithelial cells enzymically dissociated human tissue and grown to confluence in tissue culture flasks (75 cm²) with proliferation medium consisting of DMEM/Ham's F-12 and bronchial epithelial basal medium (Clonetics; Cambrex, East Rutherford, NJ) supplemented with 100 U/mL of penicillin and 100 μ/mL of streptomycin for 7 days. Cells were then trypsinized and seeded on porous polyester membranes (6∼7 X 10 ⁴ cells/membrane), in cell culture inserts (Transwell-Clear, diameter 12 mm, 0.4-μm pores; Corning, Acton, MA) coated with 100 μL of coating solution (bovine serum albumin [0.1 mg/mL; Sigma-Aldrich], type I bovine collagen [30 μ/mL; BD, St. Louis, MO], fibronectin [10 μ/mL; BD] in Laboratory of Human Carcinogenesis basal medium [Invitrogen, Grand Island, NY]) and left in a tissue culture laminar flow hood overnight. Five days later, the culture medium was removed from the upper compartment and the epithelium was allowed to differentiate by using the differentiation medium consisting of 1:1 DMEM (Invitrogen) and bronchial epithelial basal medium (Clonetics; Cambrex) with the Clonetics complements for human epidermal growth factor (0.5 ng/mL), epinephrine (5 g/mL), bovine pituitary extract (0.13 mg/mL), hydrocortisone (0.5 g/mL), insulin (5 g/mL), triiodothyronine (6.5 g/mL), and transferrin (0.5 g/mL), supplemented with 100 UI/mL of penicillin, 100 g/mL of streptomycin, 0.1 nM of retinoic acid (Sigma-Aldrich), and 10% FBS (Sigma-Aldrich) in the basal compartment.

CBF was performed as previously described.^8,10 In brief, images of beating cilia from mature cultures were visualized using a 20x objective on an inverted microscope (Leica Microsystems, Inc., Bannock-burn, IL). Image data were captured using a Model A602f-2 Basler area scan high-speed monochromatic digital video camera (Basler AG, Ahrensburg, Germany) at a sampling rate of 120 frames/s with a resolution of 640 X 480 pixels. The video images were analyzed using the Sisson-Ammons Video Analysis (SAVA) system Version 2.1 (Ammons Engineering, Mt Morris Township, MI). ¹² For each experiment, a large area of beating cilia on the ALI cultures was detected with the inverted microscope. The digital image signal was then routed from the camera directly into a digital image acquisition board (National Instruments) within a Dell XPS 710 Workstation running Windows XP Professional operating system (Microsoft, Redmond, WA). Images were captured, compressed, and stored to disk. Files were reloaded and analyzed with virtual instrumentation software highly customized to perform CBF analysis. All of the recordings for the proposed experiments were made at 200x magnification. Experiments were all performed at ambient temperature (22°C). Whole field analysis was performed with each point measured representing one cilia. For each sample, the reported frequencies represent the arithmetic means of these values, followed by standard deviations. Because the data include thousands of individual points (cilia), very small changes in CBF result in a “statistical” significance because of the high power of the analysis methods. Thus, statistical analysis of the arithmetic means derived from each culture was performed using two-tailed unpaired t-tests to ensure reproducibility. Once a stable baseline was obtained, the test solution or control solution was added at varying doses to the basolateral media or apical surface and CBF was recorded every 15 minutes for the 1st hour and then every 30 minutes for the 2nd hour. Cultures were then placed back in the incubator and analyzed again at 6 hours.

Mucociliary transport velocity was measured using 2-μm polystyrene fluorescent microspheres (0.0025% by weight in 30 μL) that were added to the apical surface of the cultures after copious washing with PBS to remove mucus clumps as previously described.^9,10 Beads were imaged using an inverted Nikon TE2000E epifluorescence microscope (20 X 0.5 NA PlanFluor objective; Nikon, Tokyo, Japan) equipped with a 12-bit QImaging camera (Q Imaging, Surrey BC, Canada) and computer running ImageJ (Wayne S. Rasband, National Institutes of Health, Bethesda, MD) and μManager. ¹³ A streak had to have a visible beginning and ending, within the field of view, to be included in the statistical analysis of the data. Either an ND4 or ND8 filter was used for bead-tracking experiments, depending on the thickness of the individual epithelial culture and resulting brightness of the beads.

Transepithelial potential (TEP) was measured using the EVOM handheld module (World Precision Instruments, Sarasota, FL) per the manufacturer's guidelines and as previously described. ¹⁴ Briefly, the apical and basolateral sides of the Costar transwell were bathed in Hank's balanced salt solution, the electrodes were inserted into the two compartments, and the TEP was taken after reaching the steady state. Myrtol^® was added to the basolateral medium at the designated concentration and TEP was reobtained 45 minutes later. TEP is reported with the basolateral as the ground side (i.e., a negative TEP equals a net lumen-negative potential).

Apical chloride permeability was measured indirectly by loading the cells with the fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium and replacing the apical chloride with NO₃⁻ as previously described. ⁹ Because Cl⁻ channels have a near equal permeability for Cl⁻ and NO₃⁻, replacement of apical Cl⁻ with NO₃⁻ results in electroneutral Cl⁻/NO₃⁻ exchange across the apical membrane. SPQ is quenched by Cl⁻ but not NO₃⁻; thus, the relative rates of SPQ fluorescence increase during NO₃⁻ substitution can be used to determine the relative rates of anion permeability. NO₃⁻ substitution experiments were performed with control Dulbecco's phosphate buffered saline containing 138 mM of NaCl for a total apical [Cl⁻] of ∼147 mM. The low Cl⁻ Dulbecco's phosphate buffered saline contained 5 mM of NaCl and 133 mM of NaNO₃ for a total apical [Cl⁻] of ∼14 mM (-10-fold reduction). SPQ was loaded by 24 hours incubation in 20 mM of SPQ at 37°C. Images were acquired with a 4′,6′-diamino-2-phenylindole filter set on an inverted Nikon TE2000E microscope (20 X 0.5 NA PlanFluor objective). SPQ fluorescence was normalized to fluorescence at time 0 (F/F _{t = 0}) as previously described. ¹⁷

Data Analysis and Statistics

Data were analyzed in SAVA, Fluoview and/or ImageJ. Statistical analyses were performed in Excel and/or GraphPad Prism (GraphPad Software, Inc., La Jolla, CA) with p < 0.05 considered statistically significant. For multiple comparisons with one-way ANOVA, Bonferroni posttest was used when preselected pairwise comparisons were performed, Tukey-Kramer posttest was used when all values in the data set were compared, and Dunnett's posttest was used when all values were compared with a control value.

Ciliary Beat Frequency

Initial experiments focused on determining effects of Myrtol^® on CBF. The sinonasal epithelium is composed of polarized cells with the apical surface exposed to the airways and the basolateral surface attached to a basement membrane and the adjacent epithelial cells. Typical nutrients are supplied to these cells via the basal surface from the vascular system. Thus, we interrogated whether there was differential effects based on the apical versus basolateral application of Myrtol^®. As shown in Fig. 1, basolateral application resulted in a dramatic increase in CBF in a dose-dependent manner with a maximum stimulation at 30 minutes of 48 ± 7%.

OPEN IN VIEWER

Figure 1

Myrtol^® stimulates ciliary beat frequency (CBF). Basolateral versus apical application of Myrtol to well-differentiated human sinonasal air-liquid interface (ALI) cultures (n = 4 ALI cultures each from different patient per concentration). All points for 0.5% basolateral application were significantly elevated compared with vehicle. For 0.1% basolateral application only 15- to 120-minute time points were significantly elevated compared with vehicle.

Basolateral application yielded persistent elevation of CBF at 0.5% for at least 6 hours (p < 0.01 for all points tested) and at 0.1% for at least 2 hours (p < 0.05). Furthermore, at the higher concentration (0.5%), apical application trended to also increase CBF but with a longer onset of action (1 hour).

Because we had previously demonstrated that nonpolypoid CRS tissue (explant) had blunted ciliary responses to purinergic, adrenergic, and cholinergic stimulation,^16,17 we wanted to make sure that ALI cultures derived from CRS patients did not show an altered ciliary response to basolateral application of Myrtol^®. Thus, cultures derived from control and CRS without polyps patients were investigated. As expected from our prior studies showing reversibility of blunted ciliary responses in CRS, the cultures derived from the CRS patients demonstrated increases in CBF comparable with cultures derived from control patients (Fig. 2). That is to say, that the source of the material, CRS versus non-CRS did not affect the cilia function in mature ALI cultures.

OPEN IN VIEWER

Figure 2

Control and chronic rhinosinusitis CRS) air—liquid interfaces (ALIs) increase ciliary beat frequency (CBF) with Myrtol. ALI cultures derived from CRS and control patients show comparable ciliary responses to Myrtol^®. Mature ALI cultures (4–5 weeks) derived from the designated patients were stimulated with 0.5% Myrtol or 0.5% ethanol on the basolateral chamber and CBF quantified at 10 an 0 minutes (n = 3 ALIs from three patients in each category). Ethanol (0.5%) was used for control cultures. A t-test was performed against the control (^***p < 0.05).

Mucociliary Transport Velocity

Recent work in rat trachea has established an increase in MCC at exceedingly small concentrations (0.01%). ⁵ Thus, we investigated the effects of basolateral application of Myrtol^® on MCC as measured by fluorescent bead transport velocity.^9,18 Although 0.1% Myrtol^® had approximately a 20% increase in CBF, we found a significantly more robust effect at this concentration on MCC. As shown in Fig. 3, 0.1% Myrtol^® applied to the basolateral surface of the human sinonasal ALI cultures yielded a 46% increase (±16%) in mucus transport velocity whereas dilution of Myrtol^® to 0.05% had no demonstrable effects on mucus transport velocity.

OPEN IN VIEWER

Figure 3

Myrtol^® stimulates mucociliary clearance (MCC) velocity; 0.1% Myrtol^® applied to the basolateral surface yielded a rapid and significant increase in MCC (n = 3).

Transepithelial Potential

Because the effects of Myrtol^® on MCC were dramatically more effective than on CBF, we questioned whether Myrtol^® may be altering the ASL viscosity via hydration. A rapid way in which to assess this possibility is with measurement of TEP. As shown in Fig. 4, 45 minutes after 0.5% Myrtol^® basolateral application, the apical surface of the cells became significantly more negative. A potential explanation is either chloride efflux or sodium absorption. However, because MCC increased (Fig. 3), we assumed this was caused by efflux of Cl⁻resulting in a transepithelial voltage that draws sodium across the epithelium via paracellular pathways, with the resulting osmotic gradient drawing water both through paracellular and through transcellular (aquaporin) pathways. The other possibility, i.e., sodium absorption, would cause dehydration of mucus and result in decreased transport velocity.

OPEN IN VIEWER

Figure 4

Myrtol^® alters transepithelial potential (TEP). Basolateral application of Myrtol alters TEP driving the apical side more negative (baseline, 2.366 ± 0.261 mV; vehicle, 2.417 ± 0.255 mV; and Myrtol^®, ±4.817 ± 0.366 mV; n = 3 per condition).

Apical Chloride Permeability

To confirm that mucociliary transport increased due to chloride efflux, we used the fluorescent dye SPQ. ⁹ In unstimulated sinonasal epithelial ALIs, replacement of apical chloride with nitrate did not result in any change in SPQ fluorescence, suggesting that resting apical chloride conductance is very low (Fig. 5 A). However, addition of Myrtol^® to the basolateral chamber caused SPQ fluorescence to increase (0.10 ± 0.02 U/min; Fig. 5 B), suggesting that apical membrane anion permeability was activated. Addition of Myrtol to the apical surface had no effect (Fig. 5, D and E). To show that the SPQ was truly measuring chloride flux, we stimulated cystic fibrosis transmembrane conductance regulator (a cAMP driven chloride channel) with forskolin, which drastically increased SPQ fluorescence (Fig. 5 F). These results (summarized in Fig. 5 G) suggest that basolateral application of Myrtol stimulates fluid secretion by activating chloride flux.

OPEN IN VIEWER

Figure 5

Myrtol stimulates chloride efflux. (A–D) Average traces of normalized SPQ fluorescence during (A and C) NO₃⁻ substitution with 0.5% ethanol or (B and D) 0.5% Myrtol. (E) Forskolin was used as a positive control. (F) Peak SPQ fluorescence (mean ± SEM) of raw traces from panels A–H (three ALIs each).

ASL Height

To confirm that the SPQ results reflected Myrtol activating epithelial fluid secretion, we directly measured sinonasal ALIs fluid secretion using techniques previously developed to measure ASL homeostasis. ¹⁹ ASL was stained with Texas red dextran before depth measurement via confocal imaging; a decrease in ASL height reflects a decrease in fluid volume whereas an increase in ASL height reflects an increase in fluid volume. Resting ASL height in human sinonasal ALIs was 11 ± 2 μm (Fig. 6 A). Cultures exposed to basolateral Myrtol exhibited significantly higher ASL after 45 minutes (24 ± 3 μm; Fig. 6 B). ASL heights are shown in Fig. 6 C.

OPEN IN VIEWER

Figure 6

Myrtol activates fluid secretion in sinonasal epithelial cells. (A and B) Confocal Z-section images of Texas red dextran–labeled airway surface liquid (ASL) from (A) 0.5% ethanol and (B) Myrtol-stimulated (45 minutes) air–liquid interfaces (ALIs; n = 3 each). (C) Graph of mean ASL heights ± SEM.