Changes in the ultrastructure of human respiratory cilia caused by the common cold were studied in 12 patients. The nasal mucosa was studied three times: on ...
Ann Otol Rhincl Laryngol 101: 1992
ULTRASTRUCTURAL CHANGES IN HUMAN NASAL CILIA CAUSED BY THE COMMON COLD AND RECOVERY OF CILIATED EPITHELIUM MARKUS RAUTIAINEN, MD
JUHANI NUUTINEN, MD
TAMPERE, FINLAND HEIKKI KIUKAANNIEMI, OULU,
KUOPIO,
MD
FINLAND
YRJO COLLAN, MD
FINLAND
'fuRKu, FINLAND
Changes in the ultrastructure of human respiratory cilia caused by the common cold were studied in 12 patients. The nasal mucosa was studied three times: on the first or second day after the beginning of symptoms, and 1 week and 3 weeks after the first biopsy. The damage was most severe at 1 week. The most remarkable finding was the loss of cilia and ciliated cells. However, the ultrastructure was usually normal, without any increase in tubular anomalies, as compared with the normal material of the previous reports. Three weeks after the beginning ofthe disease the number ofcilia and ciliated cells had increased to nearly normal. However, as a sign ofregeneration, immature short cilia (0.7 to 2.5 urnin length) were often seen. The ciliary orientation was uniform, dynein arms were normal, and there was no increase in the number of tubular anomalies. The results suggest that the impaired mucociliary function during viral infections is due to the loss of cilia and ciliated cells, rather than to ultrastructural anomalies in the cilia. The development oftubular anomalies and random ciliary orientation may require more extensive exposure to factors affecting ciliary function.
KEY WORDS -
ciliary ultrastructure, mucociliary function, respiratory infection, viral infection.
INTRODUCTION
At the time of the study there was no viral epidemic in the environment of the patients. They all had symptoms of acute common colds with acute watery rhinitis and nasal blockage for 1 to 2 days. They had a mild fever (body temperature 37.5°C to 38.5°C), but were otherwise healthy and without any history of respiratory allergy. All underwent clinical examinations by an otolaryngologist, and none had any signs of bacterial infection. After informed consent, local anesthesia was induced with cotton pieces moistened with 4% lidocaine hydrochloride with epinephrine, and a specimen for electron microscopy was taken from the middle turbinate of the nasal cavity. The first specimen was taken at the time ofdiagnosis, usually on the first or second day from the beginning ofsymptoms. The second specimen was taken I week later, and a third sample was taken 3 weeks after the first biopsy.
The common cold! is an important but complex infectious syndrome caused by a large number of different viruses.I Acute viral infections may lead to chronic respiratory diseases, and bacterial infections in the upper or lower respiratory tract are usually preceded by acute viral infections. Many kind of defects in ciliary ultrastructure have been reported in recurrent or chronic respiratory infections,3-6 in asthma," and in allergic rhinitis. s There is little doubt that viral infections of the respiratory tract can disturb mucociliary function. 9,IOExperimentally, the reduction of mucociliary clearance during viral infection has been found to be due to ciliary disorders, a decrease in the number of cilia, and decreased ciliary beat frequency. 11 This study deals with the abnormalities of the ciliary ultrastructure on nasal respiratory epithelium in patients suffering from the common cold, and the regeneration of ciliated epithelium.
During the study, no antibiotics or local nasal drugs were used. All the patients recovered in a week and had been subjectively healthy for 2 weeks before the third specimen was taken. The study plan was approved by the institutional review board for human studies at Pohja Military Hospital.
PATIENTS AND METHODS
Sixteen volunteers - nonsmoking national servicemen - were selected for this study. The patients were selected on the clinical basis of respiratory symptoms without any culture ofrespiratory viruses.
Samples for electron microscopy were fixed with I % glutaraldehyde and 4% formaldehyde fixative in 0.1 mollL phosphate buffer at 4°C for 1 week. The
From the Departments of Otolaryngology and Pathology, Kuopio University Hospital, Kuopio (Nuutinen, Rautiainen), the Department of Otolaryngology, Tampere University Hospital, Tampere (Rautiainen), the Department of Otolaryngology, Pohja Military Hospital, Oulu (IGukaanniemi), and the Department of Pathology, Turku University, Turku (Collan), Finland. REPRINTS - Iuhani Nuutinen, MD, Dept of Otolaryngology, Kuopio University Hospital, SF-70210 Kuopio, Finland.
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Fig I. Phase-contrast micrograph of nasal mucosa sampled at beginning of disease (original x187.5). Normal amount of cilia (arrowheads).
samples were then washed in veronal acetate hydrochloride buffer (pH 7.4), postfixed for 1 hour with osmium tetroxide in S-collidine buffer (pH 7.4), washed again in veronal acetate hydrochloride buffer, and stained en bloc with uranyl acetate in veronal acetate hydrochloride buffer. The samples were embedded in Epon 812 and were sectioned with a diamond knife. Ultrathin sections were stained with uranyl acetate-lead citrate 12 and examined in a Jeol JEM 100 S electron microscope in random order.
Finally, there were 12 patients in whom all three biopsy specimens were technically good enough for analysis. From the specimens the areas with the best cross sections of cilia were chosen, and electron micrographs were exposed at the primary magnification x15,OOO. The final magnification of the prints was x45,OOO. Ciliary orientation was estimated by measuring the standard deviation of the angles between the plane defined by the central tubules and a reference line, by means of a semiautomatic image
Fig 2. Electron micrograph of mucosa sampled at beginning of disease (original x45,OOO). Ciliary cross sections are uniformly normal.
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Fig 3. Phase-contrast micrograph ofmucosa sampled 1 week after onset of disease (original xI87.5). Arrowheads loss of cilia.
analyzer (mAS 1).13 RESULTS
Biopsy specimens taken at the time of the diagnosis revealed a normal number of cilia in 10 of 12 patients. The number of cilia was reduced in 2 patients. However, most specimens revealed almost normal ciliary ultrastructure with a normal amount of compound cilia, some tubular abnormalities, and occasional local ciliary disorientation (Figs 1 and 2). Most cells in the samples were ciliated, and in the ultrastructural sections we usually found a number of ciliary cross sections. In the samples taken 1 week after the beginning of symptoms, the epithelial cells were mostly without cilia or with only a few cilia (Figs 3 and 4). Only in two patients were cells found with the normal number of cilia. In the last biopsies, 3 weeks after diagnosis, the surface ofthe epithelium was usually ciliated, and the sections for electron microscopic study showed a normal number of ciliary cross sections in seven patients and a reduced number in two patients. In three patients, only a few cilia were found. Many kinds oftubular anomalies were found in the biopsy specimens at all three intervals. However, the tubular anomalies were seen only in a small percentage of cilia, and we did not find any difference between the degree ofthese anomalies in the different groups. The same number of compound cilia was found in all of the groups. Also, dynein arms were usually present and appeared to be of normal length at all three time periods. The ciliary orientation was usually very uniform in
all samples. The mean of the SD of the ciliary orientation was 30.8° (range 21.9° to 37.9°) in the first biopsy, 29.6° (range 15.7°t042.1 0) after 1 week, and 24.1° (range 17.4° to 33.2°) after 3 weeks. In some specimens taken at the time of diagnosis, we found local ciliary disorientation, but the main orientation in these samples was uniform.
In longitudinal sections ofspecimens taken 3 weeks after diagnosis, we found some cilia shorter than normal in four patients. The length ofthese short cilia varied between 0.7 urn and 2.5 um (Fig 5). The same kind of cilia were found in only one specimen taken after a week of the disease, and in no samples taken initially. DISCUSSION
During the first days ofa common cold, the changes on the nasal mucosa seemed to be slight. The degree of ultrastructural pathologic findings of the cilia was essentially the same as reported in normal controls.H The most severe damage was seen in the samples taken 1 week after the beginning of symptoms. The main finding in these samples was the loss ofcilia and ciliated cells. The same kinds ofresults were reported by Sakakura et al, II who studied ciliary injury on the nasal epithelium of chickens with scanning electron microscopy during experimentally caused Newcastle disease virus infection. Their results showed that the peak of mucociliary clearance reduction was on the seventh day of infection, and the maximum loss of cilia was on the fifth day. Also, the results ofWinther et al l S showed some sloughing of epithelial cells but no destruction of epithelial lining in patients with naturally occurring common colds. A more marked feature than the loss of cilia was local neutrophilia of
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Fig 4. Electronmicrographof nasal mucosasampled1week afteronset of disease (original x45,OOO). This section has only one cilium. There are severalbasal bodies within cell membrane (arrows).
the nasal mucosa. IS Carson et al 16 studied defects caused by acute viral respiratory infection in nasal ciliated epithelium in children. Their results s.howed a focal loss of cilia during the first week of Illness. They also reported a clearly elevated number of tubular anomalies in the cilia. In our present study the number of abnormal axonemal structures can be considered normal at different periods during the illness and the loss of cilia is markedly more pro, IS nounced than in the study of Winther et al. In the samples taken 3 weeks after the beginning of the disease, the amount of cilia and ciliated cells was normal or almost normal in 9 of 12 patients. However, as a sign ofregeneration, some short, imm.ature cilia were seen in many samples of four patients. Longitudinal sections ofthe cilia were not systematically made. However, no short cilia were seen in longitudinal sections in samples taken at the beginning of the disease. In the series of Carson et al,l6 the fine structure ofthe cilia gradually returned to normal within 6 weeks. In the organ culture of ciliated cells from patients with cystic fibrosis, the ciliogenesis was nearly complete within 1 month of injury, but there were still some short cilia. I? In this study the ultrastructure usually revealed the normal 9 plus 2 arrangement.lf and we did not notice any increase of tubular anomalies in the axonemes. It seems that
acute viral infection on the respiratory epithelium does not disturb ciliogenesis very much. On the other hand, there may be a chronic condition in which human respiratory cilia are constantly short and a cause of impaired mucociliary transport.l? An elevated number of many kinds of tubular anomalies has been reported to be associated with primary ciliary dyskinesia and many other chronic respiratory diseases.V? In the present study an elevated number of tubular anomalies in the ciliary ultrastructure could not be seen at different phases of the illness. The development of ciliary anomalies seems to require a longer exposure to infection or some other agent. Random ciliary orientation has been reported in primary ciliary dyskinesla-! and also in other chronic respiratory diseases. 22 This study shows that naturally occurring acute viral respiratory infection does not necessarily disturb ciliary orientation, and that it cannot be solely responsible for decreased ciliary function on this basis. Viral infections are known to impair mucociliary function. Experimentally induced rhinovirus infection in healthy volunteers decreased nasal mucociliary transport at 12 hours postinoculation, with a maximum depression after 3 days. The mean transport rate was slower than normal for 9 to 11 days.l? Also, an
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Fig 5. Electron micrographof mucosasampled3 weeksafter beginning of disease(originalx45,OOO). Short cilia are sign of ciliogenesis. Arrow - cilium with length of 1.5 11m.
experimental cold caused by Newcastle disease virus in chickens showed a loss of cilia and impaired mucociliary transport of the nasal mucosa.P However, mucociliary transport may occur without complete ciliation of the whole epithelial surface. Therefore, the impaired mucociliary function may partly be caused by impaired ciliary motility or coordination. Many inflammatory substances released during in-
fection are ciliostatic,24 and they' may have an influence on the viscoelastic properties of respiratory mucus. Further studies on the molecular basis of ciliary function are needed for further analysis of functional and structural changes in cilia in respiratory disease. Such studies could potentially expose vistas for more directed clinical treatment of respiratory infections.
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Rautiainen et al, Nasal Cilia & Common Cold 15. Winther B, Brofeldt S, Christensen B, Mygind N. Light and scanning electron microscopy of nasal biopsy material from patients with naturally acquired common colds. Acta Otolaryngol (Stockh) 1984;97:309-18. 16. Carson JL, Collier AM, Hu SS. Acquired ciliary defects in nasal epithelium of children with acute viral upper respiratory infections. N Engl J Med 1985;312:463-8. 17. Jorissen M, Van der Schueren B, Van den Berghe H, Cassiman J-J. In vitro ciliogenesis in respiratory epithelium of cystic fibrosis patients. Ann Otol Rhinol Laryngol 1991;100: 366-71. 18. Rautiainen M, Collan Y, Nuutinen J, Kiirjli J. Ultrastructure of human respiratory cilia: a study based on serial sections. Ultrastruct PathoI1984;6:331-9. 19. Rautiainen M, Nuutinen J, Collan Y. Short nasal respiratory cilia and impaired mucociliary function. Eur Arch Oto-
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rhinolaryngoI1991;248:271-4. 20. Canciani M, Barlocco EG, Cazzola GA, et al. The association of supernumerary microtubules and immotile cilia syndrome and defective neutrophil chemotaxis. Acta Paediatr Scand 1988;77:606-7. 21. Rautiainen M, Collan Y, Nuutinen J, Afzelius BA. Ciliary orientation in the "immotile cilia" syndrome. Eur Arch 010rhinolaryngoll990;247: 100-3. 22. Lungarella G, Fonzi L, Ermini G. Abnormalities ofbronchial cilia in patients with chronic bronchitis. Lung 1983;161:14756. 23. Sakakura Y. Changes of mucociliary function during colds. Eur J Respir Dis [Suppl] 1983;64(suppI128):348-54. 24. Motojima S. Frigas E, Loegering DA, Gleich GJ. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am Rev Respir Dis 1989;139:801-5.
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