Effects of Oxygen Radicals on Ciliary Motility in Cultured Human Respiratory Epithelial Cells

Auris·Nasus·Larynx (Tokyo) 22,178-185 (1995)

Effects of Oxygen Radicals on Ciliary Motility in Cultured Human Respiratory Epithelial Cells Masahiko YOSHITSUGU, M.D., Sinya MATSUNAGA, M.D., Yutaka HANAMURE, Markus RAUTIAINEN, M.D.,* Kazuyoshi VENO, M.D., Toshio MIYANOHARA, M.D., Shigeru FURUTA, M.D., Katsunori FUKUDA, M.D., and Masaru OHYAMA, M.D. Department of Otolaryngology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan; and Department of Otorhinolaryngology, Clinical Science, University of Tampere, Finland

*

There are few reports about direct effects of specific oxygen products on ciliary function because of their instability and reactivity. We investigated the direct effects of superoxide anion (02 -) and of hydrogen peroxide (H20 2 ) on the ciliary function of human respiratory epithelial cells, using monolayer cell cultures, high speed video analysis of frequency (CBF), amplitude (CBA), and coordination of ciliary beats and evaluating the surface structural changes of ciliated cells at the same time. 10-2 M H 20 2 decreased ciliary beat activity. The CBF was 36.5±4.4% and the CBA was 51.0±3.8% of the baseline (time=O) after 5 min (all p < 0.001). Catalase (2.ugjml) abolished the ciliotoxic effect of H 20 2 • The O2 - produced by reaction of xanthine (0.06mM)-xanthine oxidase (0.04 Ujml) caused a temporary rapid increase of 26.8 ± 1.7% in CBF and an increase of 42.5±4.1% in CBA after 15 sec (allp< 0.001). Superoxide dismutase significantly reduced these increases. Results indicated that O 2 activated ciliary function with a temporary increase in O 2 - -production. This suggests that the removal of H 20 2 from the O 2 - reaction is important in improving mucociliary clearance in excessive oxygen metabolites. Neutrophils and monocytes-macrophages in the airway epithelium present a defense against invading microorganisms. l Such antimicrobial defenses include the generation of toxic oxygen-derived species, such as superoxide (02 -), hydrogen peroxide (H 20 2 ), hydroxyl radical (OH'), and singlet oxygen e02). These oxygen products released by the phagocytes may also be involved in the pathogenesis of secretory otitis media, 2 chronic sinusitis, 3 and various lung disorders.4-6 One defense system available to the tissues to reduce such toxicity involve the release of anti-oxidative enzymes. 7 Mucociliary clearance in airway epithelium is another major defense against inhaled pathogens and microparticles. An imbalance between the amount of oxygen products and the capacity of the body to protect itself against oxidant-mediated injury may cause disease. Active oxygen species may cause mucociliary dysfunction. Although oxygen products are believed to cause ciliary dysfunction,8,9 only the direct effect of H 20 2 on ciliary function has been demonstrated. 8 Instability and high reactivity make it difficult to measure the effects of oxygen radicals in the laboratory. In this study we assessed the direct effects of O2 - and H 20 2 on the ciliary function of the human respiratory epithelial (HRE) cells, using monolayer cell cultures and a differential interference microscope equipped with a high speed video (HSV) system lO,lI Received 5 October 1994; accepted 22 May 1995. Correspondence should be addressed to: Masahiko Yoshitsugu, Department of Otolaryngology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, 890 Japan. Phone: +81-992-755410, Fax: + 81-992-648296

178 Auris·Nasus·Larynx (Tokyo) Vol. 22 (1995)

to visually analyze the ciliary beat frequency (CBF), amplitude (CBA), and the direction and coordination of ciliary beats. At the same time we evaluated surface structural changes of ciliated cells. METHODS

Cell preparation. HRE cells for five cultures were obtained from the resected maxillary mucosa of four patients who underwent a routine sinus operation for chronic sinusitis. Specimens were digested with 0.1% pronase (Type 14 protease, Sigma, St. Louis, MO, USA) in a 1 : 1 mixture of Dulbecco's modified Eagle medium and Ham's nutrient F12 (DME/F12) supplemented with penicillin (50IU/ml) and streptomycin (50,ug/ml), at pH 7.4 and kept at 4°C overnight. The dissociated epithelial cells were washed three times in DME/FI2 medium. Cells were preplated in a plastic dish at 37°C for 1 hr to reduce contamination by fibroblasts. The cell suspension was plated on collagen-coated cover glasses having a diameter of 30 mm at a density of 3,500 to 6,000 cells/cm2 , and cultured at 37°C in an atmosphere of 5% CO 2 • One day after plating, more than 80% of the cells had attached to the collagen-coated cover glasses. The culture medium consisted of a DME/FI2 medium supplemented with 10% Nu-serum, choleratoxin (10 ng/ml) , retinoic acid (l0-7 mol/liter), penicillin (50 IU/ml) , and streptomycin (50,ug/ml). The medium was changed the day after plating and every other day thereafter. Cell cultures from 2nd to 5th day after plating were used in the present study. Monitoring equipment. Ciliary motility was observed at 37°C with a differential interference microscope (Nikon, Diaphot-TMD, Tokyo, Japan) equipped with a high speed video system (nac, MHS-200, Tokyo, Japan),I0·11 using an immersion objective at 100 X magnification. The frequency, amplitude and coordination of ciliary beats were evaluated from videotapes, at a reduced speed or by examining still pictures at intervals of 0.005 sec on the monitor, 5,oooX magnification (Fig. la). By focusing on ciliated cells, the nuclei and the boundary between the cells are recognizable (Fig. 1b ). The amplitude of the ciliary beats was scored from 0 to 3 as follows: 0: amplitude resembling tremor; 1: poor but clearly recognized amplitude; 2: good amplitude; 3: very wide amplitude. Effect of H 20 2• After determining the baseline CBF and CBA (time=O), 0.1 ml of Dulbecco's modified Eagle medium (DMEM) with or without H 20 2 (at a final concentration of 10- 3 or 10- 2 M) (Santoku, Tokyo, Japan) was added by a syringe to the cell culture

Fig. 1. Cilia (A) and their ciliated cells (B) of the monolayer under microscope, photographed from the monitor at 5,OOOX magnification. N refers to the nuclei and the arrow heads point to the boundary between the cells. Bars = 5 flm. Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)

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containing 0.9 ml of DMEM containing catalase (CAT) (2tLg/ml) (Sigma) or DMEM alone. CAT is an enzyme which catalyzes the decomposition of H 20 2 to H 20 and 02. This addition was done within 5 sec to minimize physical stimulation. Ciliary movement was recorded continuously. Preparation of 02 - . Superoxide anion (02 -) produced in the xanthine (X)-xanthine oxidase (XO) reaction was assayed by the cytochrome-C reduction method. 12 A volume of 0.1 ml of DMEM with or without XO (Sigma) at a final concentration of 0.8, 4, or 8 X10- 2 Vlml was added to a reaction tube containing 0.14 ml of DMEM including cytochrome-C (1O- S M) (Sigma), X (0.06mM) (Nacalai Tesque, Inc., Kyoto, Japan) with or without the following reagents: superoxide dismutase (SOD) (60Vlml), mepacrine (10-s M), and verapamil (10- 3 M) (all obtained from Sigma). Those reagents were used as the antioxidant of 02 - , phospholipase A2 inhibitor, and Ca2+ -entry blocker, respectively. Absorbance at 550 nm was measured with a spectrophotometer (Shimadzu, UV-250, Tokyo, Japan). To investigate the absorbance induced by X- XO reaction in Ca2+ -free condition, modified Ringer solution without CaCh at pH 7.4 (NaCI, 109 mM; KCI, 3 mM; glucose, 11 mM; EGTA [ethyleneglycolbis N,N'-tetraacetic acid], 1 mM; Tris-maleate buffer, 1OmM) was used instead of DMEM. Effect of O 2-. To investigate the effect of 02 - on ciliary function, cultures of HRE cells were incubated with 1.40 ml of DMEM containing X (0.06 mM). And then 0.1 ml of DMEM with or without XO (at a final concentration of 0.8,4, or 8 X10- 2Vlml) were added to the cell cultures within 5 sec. On the other hand, 0.1 ml of DMEM with XO (at a final concentration of 4X 1O- 2 Vlml) was added to the cell cultures containing SOD (60Vlml), mepacrine (10-s M). Ciliary movement was recorded continuously. Role of CaH and O2- . To investigate the role of Ca2+ in the ciliostimulation induced by X-XO reaction, cultures were perfused with 1.4ml of the DMEM containing verapamil (10- 3 M) and X (0.06 mM). And then 0.1 ml of DMEM with XO at a final concentration of 4 X10- 2 Vlml were added to the cell culture within 5 sec. For Ca2+ -free condition, cultures were perfused with 1.4 ml of the modified Ringer solution containing X (0.06 mM) without CaCho Next, 0.1 ml of the Ca2+ -free solution with or without XO (at a final concentration of 4 X10- 2 Vlml) was added to the cell culture 1 min after exposure to Ca2+ -free solution. Statistical method. All values are expressed as mean ± SEM. Statistical analysis was performed using student's t-test. A p-value of less than 0.05 was considered statistically significant. RESULTS

Effect of H 20 2 The control study showed no significant change on CBF and CBA for 60 min. Within 2 min of exposure to 10- 2M H 20 2, we noticed a decreased CBF. CBF fell to 36.5 ±4.4% (mean ± SEM) (p < 0.00 1) of the baseline CBF after 5 min (Fig. 2). All cilia stopped moving within 10min with no obvious surface structural change noted in the ciliated cells on the monitor. CBA also decreased to 51.0±3.8% (p<0.001) of the baseline CBA after 5min (Fig. 2). Coordination and direction of the ciliary beats did not change until the ciliary movement was arrested. In contrast to what we observed with 10- 2M H 20 2, 10- 3 M H 20 2 had no significant affect on CBF and CBA within 10 min. Catalase significantly reduced the ciliotoxic effect of H 20 2 (p < 0.00 1). In the presence of catalase, CBF was 97.8 ± O. 5% of the baseline CBF 5 min after exposure to 10- 2M H 20 2. Effect of O 2Absorbance at 550 nm monitored the 02 - produced from the reaction of X and XO. XO caused a dose-dependent increase in absorbance. We noted only a small increase in absorbance 180 Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)

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LtOD,sonm (X 1O- I /min) CBF response, % § , n = 20

0.8

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Definition of abbreviations: X, xanthine; XO, xanthine oxidase; dOD, changes in optical density; CBF, ciliary beat freaquency; n, number of measured clusters of cilia. § Each value represents the mean± SEM of % change from baseline CBF 15sec after X-XOS reaction. ~p
Effects of pretreatment with reagents or CaH free. XO (4X 1O- 2 V/ml)

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2.4 26.8±1.7

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Definition of abbreviations: SOD, superoxide dismutase; MEP, mepacrine; VER, verapamiI; see Table 1. § Each value represents the mean±SEM of % change from baseline CBF 15 sec after X-XO reaction. ' p < 0.001 versus control.

when the concentration of XO was doubled from 4 X 10- 2 V/ml to 8 X 10- 2 V/ml (Table 1). An increase in absorbance induced by X-XO reaction decreased after 30sec. SOD reduced the absorbance seen in the X and XO reaction at a concentration of XO of 4 X 10- 2 V/ml (Table 2). X-XO reaction at a concentration of XO of 4X 1O- 2 V/ml caused a transient rapid increase of 26.8± 1.7% in CBF and an increase of 42.5±4.1% in CBA after 15 sec (allp
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Fig. 3. Effect of X (0.06 mM)-XO (4 X 10- 2 Vlml) reaction on frequency ( opnened circles) and amplitude (closed circles) of ciliary beat over time. The frequency and amplitude were both significantly increased immediately after exposure (p < 0.001), these increases disappeared for 2 min. n = 20 of measured cluster of cilia.

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Fig. 4. Effect of X (0.06mM)-XO (4X 10- 2 Vlml) reaction on the frequency of ciliary beat in CaH -free condition. The XXO reaction significantly reversed the decline of the frequency in the CaH -free condition (p >0.001). n =20 of measure cluster of cilia. Squares, control; circles, X-XO reaction.

except SOD (Table 2). Pretreatment with mepacrine or verapamil had no effect on the X-XO induced ciliostimulatory action (Table 2). Exposure to CaH -free solution caused a immediate decline in CBF. The X-XO reaction at a concentration of XO of 4X lQ-2U/ml reversed the decline in CaH-free condition (p
The mucociliary system defends the epithelium of the airway against inhaled pathogens and noxious microparticles. Its impairment decreases clearance and causes infection. \3 Oxidant products may cause mucociliary dysfunction8,9 and contribute to diseases. 2·6 Sources of oxidants in the airway include cigarette smoke, 14 hyperoxia, ~,9 inflammatory cells,I,6 and oxygen products within HRE cells themselves. 7 In this study we found that O2 - activated ciliary activity with a temporary increase in O2 - -production, whereas H 20 2 decreased ciliary activity. 182 Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)

Mucociliary clearance has been studied by various in vivo and vitro techniques. 13,15,16 The transmitted light method l6 is generally used for the monitoring of ciliary activity, but is mainly useful for measuring CBF. Our HSV system allowed us to visually analyze the frequency, amplitude, direction, and coordination of the ciliary beats in ciliated cells. IO,l1 Moreover, by using a monolayer cell culture on collagen-coated cover glasses, we clearly observed ciliary motility at high magnification (X 5,000) with a minimum of interference from the mucous membrane, secretory cells, and autonomic nervous system. We also examined the ciliated cells under the microscope to observe surface structural changes. Mechanical stimulation of the cell surface of cultured ciliated epithelial cells from the rabbit tracheal epithelium resulted in a transient increase. 17 Physical stimulation had a temporary stimulatory effect on the ciliary beats depressed with bacterial toxin. 18 In the present study, 0.1 ml of medium with or without agents such as H 20 2 or XO was added within 5 sec. Physical stimulation by adding the medium without agents made the ciliary function nonresponsible after 15 sec or longer, suggesting that we had excluded the effect of physical stimulation from the present study. Although free radicals are generated in most cells during normal metabolic processes, the cells protected against such injury by antioxidants. 7 Several disorders of HRE cells are caused by toxic oxygen metabolites in quantities that exceed the antioxidant capacity of the airway. 2-7 Dysfunction as well as tissue damage may occur in the airway. Airway clearance is decreased by a relatively short exposure to 95 % O2.9 This effect on mucociliary clearance appears to precede the effects on the alveolar structure. Inflammatory infiltrates have been observed in and around the ciliated epithelium in several airway diseases. 19 Oxygen products, particularly H 20 2, playa central role in the ability of the inflammatory cell to cause tissues damage. 2o H 20 2 also impairs ciliary function without causing histologic damage to the airway.9 We also found that H 20 2 rapidly decreased ciliary activity with no obvious surface structural change of the cell on the monitor. O2- has been implicated in postischemic tissue injury.21 There have been no reports regarding the direct effect of O2- on ciliary function. Conversion of xanthine to uric acid by xanthine oxidase generates O2- . We used the X and XO reaction to produce O2- . We found that the X-XO reaction brought about a rapid temporary increase in ciliary activity. The production of O2- from the X- XO reaction measured spectrophotometric ally, related to ciliary response. A decline in the ciliostimulatory action of X- XO reaction without falling below the baseline for 60 min, induced by pretreatment with SOD, proved the ciliostimulative effect of O2- from the X-XO reaction. Our results indicated that temporary products of O2- activate ciliary function, and thus may serve in part as a host defense mechanism in improving mucociliary clearance. Ciliostimulation is associated with the movement of CaH in the oviduct. 22,23 Low concentrations of Ca channel blocker do not always completely block CaH influx into cell. 24 The frequency response of ciliary beats to mechanical stimulation was unaltered in the presence of 100,uM verapamil. However, increasing the verapamil concentration to 1 mM greatly reduced the frequency response. 17 The presence of a high concentration of CaH -entry blocker (1 mM verapamil) and a CaH or a CaH -free condition did not effectively inhibit the increase in CBF produced by O2-, Our results suggest that O2- induced ciliostimulation may be independent of CaH influx into the cell. Reactive oxygen species can provoke the release of arachidonic acid from membrane phospholipid. 25 Also, arachidonic acid can be synthesized by cultured airway epithelial cells. 26 Prostaglandins and leukotrienes can increase CBF in airway epithelial cellsY Mepacrine (10- 5 M), phospholipase A2 inhibitor, reduces the increase in CBF elicited by angiotensin through prostaglandin release in rabbit cultured tracheal epithelium. 28 We found that 10- 5M mepacrine did not inhibit the increase in CBF elicited by O2- from X-XO reaction. Our results suggested Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)

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that the ciliostimulatory action of O2- may be independent of the arachidonic acid released from the airway epithelial cells, and also may be the direct effect on the cilia. The neutrophils and macrophages produce toxic oxygen-derived species. 1 Reactive products of O2 metabolism can be formed from reaction of O2-. O2- is not always itself the species that cause host defense or tissue damage, but is the precursor of a more potent oxidant, whose generation depends on the simultaneous presence of H 20 2:7 O2- + H202~OH- +OH' + 102 Neutrophils can kill pulmonary endothelial cells by producing H 20 2.20 The OH· resulting from the interaction of O2- and H 20 2 is also lethal to the cell. 29 The rapid removal of H 20 2 by using catalase to bring about the reaction of H 20 2 to H 20 and O2 would therefore diminish the production of the highly reactive OH· radical and reduce tissue damage. Catalase protects the cell against the ciliotoxic effect ofH 20 2•9 In the present study, we found that catalase abolished the ciliotoxic effect of H 20 2. In conclusion, results indicate that O2 - activate ciliary activity with a temporary increase in O2 - productions. The removal of H 20 2 appears to be important in improving mucociliary clearance as well as reducing tissue injury in the excessive oxygen metabolites. The authors thanks Ms. S. Katahira for expert technical assistance. REFERENCES

1. Babior M: Oxygen-dependent microbial killing by phagocytes. N Engl J Med 298:659-668, 1978. 2. Ovesen T, Borglum JD: Superoxide dismutase in middle ear fluid from children with secretory otitis media. Acta Otolaryngol (Stockh) 112:1017-1024,1992. 3. Matsunaga S, Hoh K, Babazono M, et al: Effects of Shin'i-seihai-to and Sho-seiryu-to on production of active oxygen species by neutrophils. Pract Otol (Kyoto) 85:1975-1980, 1992. 4. Repine JE: Scientific perspectives on adult respiratory distress syndrome. Lancet 339:466-469, 1992. 5. Martin WJ, Gadek JE, Hunninghake GW, et al: Oxidant injury of lung parenchymal cells. J Clin Invest 68:1277-1288, 1981. 6. Kroegel C, Yukawa T, Dent G, et al: Stimulation of degranulation from human eosinophils by platelet-activating factor. J Immunol 142:3518-3526, 1989. 7. Fridovich I: The biology of oxygen radicals. Science 201:875-880, 1978. 8. Burman WJ, Martin WJ: Oxidant-mediated ciliary dysfunction: possible role in airway disease. Chest 89:410-413, 1986. 9. Sackner MA, Landa J, Hirsch J, et al: Pulmonary effects of oxygen breathing: a 6-hour study in normal men. Ann Intern Med 82:40-43, 1975. 10. Rautiainen M, Matsune S, Shima T, et al: Ciliary beat of cultured human respiratory cells studied with differential interference microscope and high speed video system. Acta Otolaryngol (Stockh) 112:845-851, 1992. 11. Yoshitsugu M, Rautiainen M, Matsune S, et al: Effect of exogenous ATP on ciliary beat of human ciliated cells studied with differential interference microscope equipped with high speed video. Acta Otolaryngol (Stockh) 113:655-659, 1993. 12. McCord JM, Fridovich I: Superoxide dismutase: An enzymic function for erythrocuprein (hemocuprein). J BioI Chem 244:6049-6055, 1969. 13. Lourenco RV, Klimek MF, Borowski CJ: Deposition and clearance of 2/1 particles in the tracheobronchial tree of normal subjects--smokers and nonsmokers. J Clin Invest 50:1411-1420, 1971. 14. Nakayama T, Kaneko M, Kodama M, et al: Cigarette smoke induces DNA single-strand breaks in human cells. Nature 314:462, 1985. 15. Mercke V, HaKansson H, Toremalm NG: A method for standardized studies of mucociliary activity. Acta Otolaryngol (Stockh) 78:118-123, 1974. 184 Auris'Nasus'Larynx (Tokyo) Vol. 22 (1995)

16. Yager J, Chen TM, Dulfano MJ: Measurement of frequency of ciliary beats of human respiratory epithelium. Chest 73: 627-633, 1978. 17. Sanderson MJ, Dirksen ER: Mechanosensitivity of cultured ciliated cells from the mammalian respiratory tract: implications for the regulation of mucociliary transport. Proc Natl Acad Sci USA 83:7302-7306, 1986. 18. Rautiainen M, Yoshitsugu M, Matsune S, et al: Effect of exogenous ATP and physical stimulation on ciliary function impaired by bacterial endotoxin. Acta Otolaryngol (Stockh) 114:337-340, 1994. 19. Wanner A: Clinical aspects of mucociliary transport. Am Rev Respir Dis 116:73-125, 1977. 20. Martin WJ: Neutrophils kill pulmonary endothelial cells by a hydrogen-peroxide-dependent pathway. Am Rev Respir Dis 130:209-213, 1984. 21. McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312:159-163, 1985. 22. Eckert R, Murakami A: Calcium dependence of ciliary activity in the oviduct of the salamander necturus. J Physiol 226:699-711, 1972. 23. Verdugo P: CaH -dependent hormonal stimulation of ciliary activity. Nature 283:764-765, 1980. 24. Lee KS, Tsien RW: Mechanism of calcium channel blockade by verapamil, D600, diltiazen and nitrendipine in single dialysed heart cells. Nature 302:790-794, 1983. 25. Martin EH, Cook HW, Lands WEM: Prostaglandin biosynthesis can be triggered by lipid peroxides. Arch Biochem Biophys 193:340-345, 1979. 26. Eling TE, Danilowicz RM, Henke DC, et al: Arachidonic acid metabolism by canine tracheal epithelial cells. J BioI Chern 261:12841-12849, 1986. 27. Wanner A, Maurer D, Abraham WM, et al: Effects of chemical mediators of anaphylaxis on ciliary function. J Allergy Clin Immunol 72:663-667, 1983. 28. Kobayashi K, Tamaoki J, Sakai N, et al: Angiotensin stimulates airway ciliary motolity in rabbit cultured tracheal epithelium. Acta Physiol Scand 138:497-502, 1990. 29. McCord JM, Day ED: Superoxide-dependent production of hydroxyl radical catalyzed by ironEDTA complexes. FEBS Lett 86:139-142, 1978.

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