A new approach to pharmacological effects on ciliary beat frequency in cell cultures--exemplary measurements under Pelargonium sidoides extract (EPs 7630).
The ciliary beat frequency (CBF) is an important parameter of the defence mechanism of the mucociliary system. We present a new method to determine pharmacological effects on CBF in vitro. Ciliated cell cultures of human nasal epithelium were obtained from partial resection of hyperplastic inferior turbinates in rhinosurgery. An adherent monolayer culture of ciliated cells was present after 10 days in vitro. This study exemplary examines, if a special extract from the roots of Pelargonium sidoides (EPs 7630) has an effect on the CBF in vitro. The influence of three concentrations of the extract (1, 30, 100 [micro]g/ml) was tested. EPs 7630 significantly and concentration-dependently increased CBF to 123% at 30 [micro]g/ml and to 133% at 100 [micro]g/ml compared to the equilibration phase (100%). After rinsing with extract-free medium the CBF of cultured cells returned to nearly the normal range. In future, drug manipulation of the CBF by local application of rhinologics could be a new therapeutical concept in the treatment of upper airway diseases.
[c] 2004 Elsevier GmbH. All rights reserved.
Keywords: Cell culture; Human nasal epithelium; Ciliated cells; Ciliary beat frequency; Pelargonium sidoides root extract; EPs 7630
The nasal cavity is the common entrance for inhaled air to the respiratory tract. Important protective functions of the inner nose are heating, humidifying and cleaning the gas. The mucociliary system is an important defence mechanism for cleaning the air of bacteria and foreign particulate matter. It is a well-coordinated system consisting of airway secretory cells that produce a sol and gel fluid layer on the airway surface and ciliated cells that propel the mucus towards the nasopharynx (Deitmer, 1989, 1996).
The most important parameter of the mucociliary system for the purpose of cleaning is the ciliary beat frequency (CBF) of ciliated cells (Boek et al., 2002; Braverman et al., 1998; Duchateau et al., 1985). Inflammatory noxa of viral or bacterial infection in the upper respiratory system can cause damage of the mucociliary system, causing congestion of mucus secretion in the nose and the paranasal sinuses. Consequences are post nasal drip and superinfection of nasal discharge. Medical treatment of mucociliary clearance by pharmacological increase of CBF could be a basic therapeutic approach by eliminating the congestion of mucus secretion. In case of acute rhinitis, sinusitis and bronchitis it could abbreviate the course of these disorders or may even stop the diseases at an early stage. Also pathology of chronic sinusitis could be clearly alleviated. CBF cannot be measured under in vivo conditions. We present a new method to determine pharmacological effects on CBF in vitro and exemplary measurements under Pelargonium sidoides extract (EPs 7630).
Clinical trials show that symptoms of acute bronchitis like cough, sputum and dyspnea are abated under treatment with a radix extract of P. sidoides (EPs 7630) (Blochin et al., 1999). Responsible for this effect are antibacterial and immunomodulatory characteristics of the extract. Experimental research points to an inhibition of growth of gram positive and gram negative bacteria (Kayser and Kolodziej, 1997). Other studies suggest an activation of unspecific immune defence mechanisms (e.g., induction of iNO, release of TNF-alpha) (Kayser et al., 2001).
It was the aim of the present study to examine, if increasing concentrations of EPs 7630 extract have an effect on CBF of cultured human nasal epithelial cells. Our special developed equipment provides standardized testing conditions.
Material and methods
Origin and isolation of epithelial cells
Respiratory mucosa for cell culturing was obtained from partial resection of hyperplastic inferior turbinates at rhinosurgery. The patients had preoperatively given their informed consent.
The tissue was placed for less than 5 h into DMEM (Sigma) supplemented with antibiotic/antimycotic solution [penicillin (200 U/ml), streptomycin (200 [micro]g/ml) and amphotericin B (0.5 [micro]g/ml)]. To minimize the risk of contamination, the tissue was immersed in 1% iodine polyvidone (Betaisodona[R]) in PBS for 3 min and was afterwards rinsed three times in PBS. After washing the specimens were immersed in 0.01% protease from Streptomyces griseus (Sigma) in serum free culture medium for 3-4 h at 34 [degrees]C. In order to stop enzyme activity, the tissue was transferred into culture medium (see below) supplemented with 10% fetal calf serum (FCS) (Gibco). By gentle agitation with a scalpel, epithelial cells were released as single cells, cell clusters and larger mucosal sheaths into the medium. After filtration through a stainless steel sieve, the cell suspension was centrifuged at 150 g for 10 min and resuspended in serum free culture medium (Neugebauer et al., 1996).
The culture medium was a mixture of DMEM and Ham's F12 (Sigma), supplemented with insulin (1 [micro]g/ml), transferrin (1 [micro]g/ml), hydrocortisone (0.5 [micro]g/ml), retinoic acid (10 ng/ml), L-glutamine (3.2 mM), and 1.25% antibiotic/antimycotic solution.
Submersion cell culture
For incubation of cells tissue culture dishes (EASY GRIP[TM], Falcon[R]) were used. The bottom of the culture dishes was coated with extracellular matrix (ECM) molecules of the basal lamina.
The following stock solutions of ECM-molecules were used for coating: 50 [micro]l collagen IV (collagen IV (0.5 mg/ml) in 0.25% acetic acid), 10 [micro]l laminin and 1 [micro]l heparan sulphate proteoglycan (HSPG) were added to 1 ml PBS in each culture dish and incubated at 34 [degrees]C for 24 h. The ECM-coating solution was removed and the dishes were washed and filled with the culture medium. The resuspended cells were seeded in submersion at high density in tissue culture dishes and kept in an incubator (humidified atmosphere, 10% C[O.sub.2], 34 [degrees]C). Every 3 days, the culture medium was changed (Neugebauer et al., 2003).
Measurement of ciliary beat frequency
Ciliary beat frequency of cultured cells was measured in a custom-made test facility (Fig. 1). An inverted research microscope (DM IRB, Leica) with a heated stage (HT 200, Minitub) was used. A colour video camera (CCD-Iris/RGB, Sony) transmitted the microscopic picture to a monitor. The medium reservoir for a maximum of eight different types of media was valve-controlled. The tempered culture medium (Temperature-Controller HT 1.2, Vetec) was continuously changed pulseless (Masterflex digital console drive, Cole-Parmer Instruments) with a rate of 1 ml/min. Thus, different concentrations of EPs 7630 could be completely replaced with new culture medium within 7 min. Temperature and pH were constantly controlled in the cell cultures. Each experiment was recorded on video tape. The CBF of single cells was measured according to the following log (Table 1).
CBF was determined optically (custom made) from the monitor and computerized with the help of an FFT-Analyzer (CF-350 Z, Ono Sokki).
To be sure, that the CBF of the cell culture is not influenced by the measuring period, consistency of measurements was evaluated in a pilot study.
[FIGURE 1 OMITTED]
EPs 7630, a special aqueos-ethanolic, not standardized extract (11% m/m) from the roots of P. sidoides (Kolodziej et al., 2002) was provided by Dr. Willmar Schwabe GmbH and Co. KG, Karlsruhe, Germany. Powdered extract (50 mg) was solved in 10 ml distilled water with 10% dimethyl sulfoxide (DMSO), mixed by vortexing and filtrated sterilely.
This concentrated stock solution was diluted with culture medium for final extract concentrations of 1 [micro]g/ml (0.002% DMSO), 30 [micro]g/ml (0.06% DMSO) and 100 [micro]g/ml (0.2% DMSO).
An adherent monolayer culture of ciliated cells was present after 10 days in vitro (Fig. 2). There was no influence of DMSO (max. 0.2%) on the CBF in preliminary tests.
The CBF over the time was constant for up to 180 min in comparison to the equilibration phase (7.3[+ or -]1.4 Hz=100%) (Fig. 3) and showed no significant difference (n=7; 29 single cells, two-sided t-test, p < 0.05). The CBF during the equilibration period of the EPs 7630 study (6.9 [+ or -] 1.4 Hz =100%) was not significantly different from the mean values measured during the pilot study on time consistency.
Increasing concentrations of EPs 7630 stimulated the CBF of the cultured cells. While 1 [micro]g/ml extract had nearly no effect (7.2 [+ or -] 1.6 Hz = 103%), 30 [micro]g/ml significantly increased CBF to 8.4 [+ or -] 1.7 Hz (= 123%) and 100 [micro]g/ml to 9.1 [+ or -] 1.8 Hz (=133%) (Fig. 4). After thorough rinsing with extract-free medium (0 [micro]g/ml) the CBF of cultured cells returned roughly to the normal range and was not significantly different from the equilibration period.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Our test facility provides a new approach to pharmacological effects on CBF in human cell cultures under standardized testing conditions. The presented exemplary measurements demonstrate that CBF is stimulated significantly by P. sidoides extract EPs 7630. This stimulation of CBF has not been described yet and represents--beside the antibacterial and the immunomodulatory activity (Kayser and Kolodziej, 1997; Kayser et al., 2001)--a possibly new therapeutic effect of this extract.
The pharmacological active principle of the nonstandardized EPs 7630 extract responsible for the effect on CBF is yet unknown, so that follow up studies will be necessary to provide further information about the ciliostimulatory ingredients. First of all, research should be conducted to evaluate if a cAMP-mediated stimulation of CBF similar to those induced by [[beta].sub.2]-agonists and prostaglandins is involved or if the effects are due to an upregulation of cGMP as has been described for iNO and agonists on muscarinic receptors (Yang et al., 1996; Zagoory et al., 2002).
[FIGURE 4 OMITTED]
Although stimulation of CBF by EPs 7630 under cell culture conditions is statistically significant, follow up examinations have to prove if this in vitro effect also is evident under in vivo conditions and if it promotes mucociliary clearance. While CBF is assumed to be one of the main parameters (Boek et al., 2002; Braverman et al., 1998; Duchateau et al., 1985), other parameters like quality and quantity of the nasal mucus under pathological conditions of inflammation and infection could eliminate some of the positive effects of mucociliary clearance.
The present results suggest that the local application of EPs 7630 close to the nasal mucosa might promote CBF of ciliated cells. Main ingredients of EPs 7630 are cumarins, procyanides, tannins and phenol carboxylic acids. At present, extract of P. sidoides is only available for systemic application. Local application could be limited by a moderate astringent effect of tannins. As a first step, a clinical trial on healthy test persons could clarify if oral or nasal application of EPs 7630 significantly abbreviates the indigo-carmine-saccharin-test (Anderson et al., 1974; Braat et al., 1995; McMahon et al., 1997; Stanley et al., 1985; Van de Donk et al., 1982). In a second step, clinical trials might show if stimulation of CBF indeed abbreviates the course of acute sinusitis or if it alleviates disorders such as chronic sinusitis. In future, pharmacological manipulation of the CBF by local or systemic application of rhinologics could be a new therapeutical concept in the treatment of upper airway diseases.
Table 1. Log for measurement of CBF with different concentrations of EPs 7630 extract Concentration Equilibration 1 [micro]g/ml 30 [micro]g/ml At time (min) 0, 20, 40 60, 80 100, 120 Concentration 100 [micro]g/ml 0 [micro]g/ml At time (min) 140, 160 180, 200
This study was supported by the Jean-Uhrmacher-Foundation, Cologne, Germany and W. Spitzner Arzneimittelfabrik GmbH, Ettlingen, Germany.
Received 13 October 2003; accepted 20 November 2003
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P. Neugebauer (a,*), A. Mickenhagen (a,b), O. Siefer (a,b), M. Walger (a,b)
(a) ENT-Department, University of Cologne, Cologne, Germany
(b) Jean-Uhrmacher-Institut fur klinische HNO Forschung, University of Cologne, Cologne, Germany
*Corresponding author. Tel.: + 2214784760; fax: + 2214784793.
E-mail address: firstname.lastname@example.org (P. Neugebauer).