Abstract
The increased susceptibility of the elderly to lower respiratory tract infection cannot be fully explained. Although mucociliary clearance, which is affected by ciliary beating and ultrastructure, plays a crucial role in the defense of the airways against inhaled microbes, little is known of the effects of aging on these parameters. We studied the nasal mucociliary clearance (NMCC) time, ciliary beat frequency, and ultrastructure of respiratory cilia in a cohort of healthy volunteers (age range 11 to 90 yr). Ciliary beat frequency of ciliated nasal epithelial cells was obtained via an established photometric method, and NMCC time was measured with the saccharine test. There was a correlation of ciliary beat frequency (r = − 0.48, p = 0.0001) and NMCC time r = 0.64, p < 0.001) with increasing age. Transmission electron microscopy revealed an increase in the percent of subjects exhibiting microtubular disarrangement and single central microtubules with aging (p = 0.002 and p = 0.005, respectively). Subjects older than 40 yr of age had significantly slower ciliary beat frequency, higher percent of ciliary cross-sections displaying single tubules, and longer NMCC time than their younger counterparts (p < 0.05). These findings may help explain the frequent occurrence of respiratory infection in the elderly.
The respiratory epithelium is essential for defense of the airways against inhaled pathogens. When bacteria or particles of less than 0.5 μm in size reach the lower respiratory tract, they frequently adhere to surface mucus that is conveyed to the nasopharynx and periodically swallowed. The efficacy of this mucociliary clearance depends on the function of healthily beating respiratory cilia (1, 2). These cilia may have functional defects when their beating becomes abnormally slow or uncoordinated (i.e., dyskinetic). Numerous ultrastructural defects or anomalies of the classical “9+2” structure of respiratory epithelial cilia have been associated with ciliary dyskinesia (Figure 1). For mucociliary clearance to be effective, neighboring cilia have to beat in a coordinated fashion and in the same direction. The direction of ciliary beating, which is determined by the orientation of the central microtubules of the cilia, can be assessed with transmission electron microscopy (TEM) (3). Recently, the orientation of central ciliary microtubules in some patients with chronic bronchial sepsis was found to be abnormal despite normal ciliary beating and otherwise normal ultrastructure (3). Abnormal ciliary function, which could result from an abnormal ultrastructure or occur de novo, would lead to chronic sepsis of the upper and lower respiratory tracts (1, 2, 4, 5).
Fig. 1. Schematic diagram showing cross-sectional ultrastructure of a cilium. There are nine peripheral microtubules and two central microtubules, which are arranged in an orderly manner. Central microtubules from neighboring cilia are normally oriented in the same plane and in parallel.
[More] [Minimize]Although mucociliary clearance might decline with aging (6), little is known about the effect of increasing age on ciliary beat frequency or the orientation of ciliary central microtubules, which actually determine the efficiency of ciliary actions. However, a decline in mucociliary clearance could theoretically lead to increased susceptibility to and severity of lower respiratory tract infection, which is known to affect the elderly (7, 8). Although physiologic, structural, and other changes occur in the senescent lung, there are few clues to the reasons for the predisposition of the elderly to respiratory infections (9-12). Although ciliary defects or impairment in mucociliary clearance might account for the high incidence of lower respiratory tract infection in the elderly, few conclusive data exist on the effect of aging on ciliary function (13-16). We therefore studied the effects of aging on the function and ultrastructure of respiratory cilia.
Nonsmoking Chinese subjects in stable health were recruited into the study at Queen Mary Hospital of the University of Hong Kong through verbal consent. The exclusion criteria included respiratory or nasal symptoms within the preceding 2 wk, active infections, known primary ciliary dyskinesia, bronchiectasis, valvular heart diseases, and bleeding diatheses. The study protocol had approval from the institutional ethics committee of the University of Hong Kong.
Nasal epithelium was obtained without anesthesia from the inferior turbinate of subjects by using a cytology brush (17), and was suspended in 0.5 ml of Medium 199 (Flow Laboratories, New York, NY). The suspension was sealed within a rim of high-density petroleum gel between a glass cover slip and a microscope slide. This preparation was examined with a Leica DM LB phase-contrast microscope equipped with a warm stage maintained at 37° C (Leitz, Wetzlaar, Germany). The beating cilia, examined at a magnification of ×100, were positioned to interrupt light emitted through a slit, and the frequency of interruption was converted into ciliary beat frequency (Hz) by an MPV-COMBI photomultiplier (Leitz) and a custom-made digital converter (18). Ciliary beat frequency was assessed at 10 different sites in the respiratory cell suspension, which were at least one microscope field (×100) apart from each other. The mean value for these 10 sites was taken as the ciliary beat frequency of the subject. The percent of ciliary cross-sections displaying an ultrastructural defect was noted for each patient.
The ciliary suspension was fixed in 2.5% cacodylate-buffered glutaraldehyde (pH 7.2) and postfixed in 1% osmium tetroxide. This was followed by standard serial dehydration through alcohols and embedding in araldite. An ultrathin section (70 to 90 nm) through the central portion of each specimen was examined with TEM at a magnification of ×3,000. During TEM, a systematic evaluation of the ciliary cross-section was performed to assess the quantity and arrangement of the central and peripheral microtubules. This included assessment for the presence of microtubular disarrangement (disruption of the spatial arrangement of microtubules), extra tubules (more than “9+2” microtubules), and single tubules (instead of paired microtubules). Only specimens that had more than 10 distinctly visible and complete ciliary cross-sections were analyzed.
The orientation of the ciliary central microtubules was also assessed for each patient as described previously (3, 18). Briefly, sections of cilia were taken from each randomly chosen ciliated epithelial cell for further examination by TEM. An image of these cilia was electronically captured and processed with an image analysis system (Improvision, London, UK). For each cilium, a line was drawn electronically through the central pairs of microtubules. The angle made by each of these lines with the horizontal axis was then measured. The standard deviation (SD) of these angles for the ciliary central microtubules from each of the epithelial cells was calculated. A mean ± SD value was obtained from all the epithelial cells for each patient, which represented an index of ciliary central microtubular orientation (3). By convention, only TEM specimens with more than 20 cilia captured in full cross-section, and obtained from at least six different epithelial cells, were analyzed (18).
NMCC time was assessed with the saccharine test as described previously (19). Briefly, a saccharine tablet (1 × 1 × 1 mm3) was inserted under direct vision with a pair of Tilley nasal dressing forceps into the medial aspect of the inferior turbinate of one nasal cavity. The time from saccharine placement until the subject, who sat forward quietly with the head bent forward, reported the first sensation of a sweet taste was measured to the nearest minute. The result was expressed as NMCC time, which negatively reflects the efficiency of NMCC, and is normally between 20 and 60 min (19). The saccharine test was performed from 1 to 3 mo after the initial nasal brushing to allow time for healing. In six cases, the test was performed 4 mo after the initial nasal brushing so that the subjects were in stable health, as described earlier.
Data were calculated as mean, standard deviation, SD and range. Pearson's correlation was used to examine the relationship between ciliary beat frequency, NMCC time, and age, as well as the mean ± SD of orientation angles and age. The subjects were divided into subgroups according to age; the relationship between frequency of various microtubular ultrastructural abnormalities and age was analyzed with the chi-square test for trend. A value of p < 0.05 was considered to indicate statistical significance. The analysis was performed with SAS software (version 8; SAS Institute Inc., Cary, NC).
Ninety subjects (47 males and 43 females) were recruited between June 1998 and May 1999. The age of the subjects (mean ± SD) was 51.7 ± 23.2 yr (range: 11 to 90 yr). The subjects' comorbidities and medications are shown in Table 1. There was no significant difference in the age of male and female subjects (51.4 ± 23.1 yr and 51.1 ± 23.1 yr, respectively; p = 0.12).
| Age Group (yr) | Patients (n) | Female Sex n (%) | Other Significant Medical Conditions (no. patients) | Medications (no. patients) | ||||
|---|---|---|---|---|---|---|---|---|
| 11–20 | 2 | 0 | 0 | 0 | ||||
| 21–30 | 26 | 9 (34.6) | 0 | 0 | ||||
| 31–40 | 15 | 7 (46.7) | 0 | 0 | ||||
| 41–50 | 6 | 3 (50.0) | 0 | 0 | ||||
| 51–60 | 10 | 5 (50.0) | IHD (1), epilepsy (1), DM (1) | Antianginal (1), antiepileptic (1) | ||||
| 61–70 | 6 | 4 (66.7) | IHD/HT (4), CVD (3), DM (2), PU (1) | Antianginal (4), hypoglycemic (2) | ||||
| 71–80 | 13 | 7 (53.8) | IHD/HT (16), CVD (9), DM (7), | Antianginal (11), H2-antogonist (3), | ||||
| PU (3), renal impairment (2), old TB (2), | hypoglycemic (2) | |||||||
| 81–90 | 12 | 8 (66.7) | IHD/HT (9), DM (5), CVD (4), | β-antagonists (9), H2-antagonist (2), | ||||
| PU (2), old TB (2), renal impairment (1) | hypoglycemic (2) |
Light-microscopic assessment was done on ciliary specimens from the entire cohort of 90 subjects (Table 2). The ciliary beat frequency was 13.0 ± 1.7 Hz (range: 9.1 to 17 Hz) and was identical for both male and female subjects (13.0 ± 2.1 Hz and 13.0 ± 1.8 Hz, respectively; p = 0.97). Some subjects had slowly beating cilia, but none had dyskinetic or immotile cilia. No other light-microscopic abnormalities were detected. Patients older than 40 yr of age had a significantly lower ciliary beat frequency (12.2 ± 1.7 Hz) than their younger counterparts (13.9 ± 1.4 Hz) (p < 0.001).
| Age Group (yr) | No. Subjects | Ciliary Beat Frequency (Hz) | No. Subjects | Percent of Subjects Showing Ultrastructural Abnormalities | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Disarrangement* | Extra- Tubules* | Single Tubules* | ||||||||||||||
| Mean | SD | Range | ||||||||||||||
| 11–20 | 2 | 13.6 | 0.1 | 13.5–13.6 | 2 | 0 | 0 | 0 | ||||||||
| 21–30 | 26 | 13.8 | 1.4 | 10.6–17.0 | 26 | 15.4 | 11.5 | 11.5 | ||||||||
| 31–40 | 15 | 14.2 | 1.4 | 12.3–17.0 | 15 | 6.7 | 26.7 | 6.7 | ||||||||
| 41–50 | 6 | 12.4 | 1.1 | 10.6–13.9 | 6 | 50 | 33.3 | 16.7 | ||||||||
| 51–60 | 10 | 12.4 | 1.4 | 9.6–14.6 | 8 | 25 | 25 | 37.5 | ||||||||
| 61–70 | 6 | 13.0 | 1.7 | 10.5–14.7 | 5 | 20 | 0 | 40 | ||||||||
| 71–80 | 13 | 12.1 | 1.9 | 9.5–16.0 | 8 | 50 | 37.5 | 25 | ||||||||
| 81–90 | 12 | 13.0 | 2.3 | 9.1–15.6 | 5 | 80 | 60 | 60 | ||||||||
Only 75 and 39 subjects, respectively, had TEM specimens adequate for assessment of microtubular ultrastructures and ciliary central microtubular orientation angle (Table 2). Altogether, a mean of 90.3 ± 87.0 (range: 10 to 419) ciliary cross-sections were examined for the cohort. Of these, 25%, 20%, and 20% exhibited microtubular disarrangement, extra tubules, and single tubules, respectively. The percents of ciliary cross-sections exhibiting microtubular disarrangement (overall: 3.1 ± 1.9; male subjects: 3.0 ± 2.0; and female subjects: 3.1 ± 2.0%; p = 0.92), extra tubules (3.7 ± 6.4, 3.5 ± 6.6, and 3.8 ± 6.5%, respectively; p = 0.92), and single tubules (2.2 ± 1.7, 2.4 ± 1.8, and 2.1 ± 1.7%, respectively; p = 0.52) were not significantly different between the two sexes. There was a significant trend toward the percent of subjects displaying ciliary ultrastructural abnormalities (i.e., microtubular disarrangement, p = 0.002; extra tubules, p = 0.06; and single tubules, p = 0.005) increasing with advancing age. Patients over the age of 40 yr had a significantly higher percent of ciliary cross-sections that displayed single tubules (2.5 ± 1.8% versus 0.8 ± 0.3%; p = 0.02), but not microtubular disarrangement (3.4 ± 2.1% versus 2.4 ± 1.5%, p = 0.19) or extra tubules (4.2 ± 8.2 versus 3.1 ± 2.4%, p = 0.62) than did their younger counterparts.
The ciliary central microtubular orientation angle was 12.7 ± 3.9 degrees (range: 5.6 to 22.3 degrees). Male and female subjects had similar central microtubular orientation angles (12.5 ± 4.2 degrees and 13.0 ± 3.7 degrees, respectively; p = 0.72). There was no significant difference in the ciliary central microtubular orientation angles between subjects over and under 40 yr of age (13.2 ± 4.0 degrees versus 12.1 ± 3.3 degrees, respectively; p = 0.37).
Of the 90 subjects in the study, 43 successfully completed the saccharine test (Table 3). Twenty-nine subjects were unable to perform the saccharine best because of inability to maintain posture (n = 17) or repeated sneezing after insertion of the saccharine tablet (n = 12). Eighteen subjects declined to proceed despite repeated invitations to participate. There was no significant difference between male (n = 24) and female subjects (n = 19) in NMCC time (12.8 ± 5.6 min and 12.5 ± 5.9 min, respectively; p = 0.87). There was a significant difference between the NMCC time for subjects under and over the age of 40 yr (9.3 ± 5.2 min versus 15.4 ± 5.0 min, respectively; p < 0.001).
| Age group (yr) | No. Subjects | Subjects Who Completed Saccharine Test n (%) | NMCC time (min) | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Mean | SD | Range | ||||||||
| 11–20 | 2 | 1 (50%) | 7.9 | NA | NA | |||||
| 21–30 | 26 | 12 (46.2%) | 9.8 | 5.1 | 2–20 | |||||
| 31–40 | 15 | 7 (46.7) | 9.7 | 5.0 | 9–20 | |||||
| 41–50 | 6 | 4 (66.7%) | 9.7 | 5.0 | 6–17 | |||||
| 51–60 | 10 | 5 (50%) | 10.1 | 5.2 | 8–20 | |||||
| 61–70 | 6 | 3 (50%) | 12.6 | 5.9 | 6–9 | |||||
| 71–80 | 13 | 8 (61.5%) | 12.1 | 5.9 | 16–20 | |||||
| 81–90 | 12 | 3 (25%) | 12.6 | 6.0 | 17–20 | |||||
There was wide intersubject variation in ciliary beat frequency. In this cross-sectional study, slower beat frequency was associated with advancing age (r = −0.48, p = 0.0001; Figure 2A). However, there was no significant difference in ciliary beat frequency between subjects with and without microtubular abnormalities. On the other hand, there was a weak correlation between ciliary orientation angle and age (r = 0.28, p = 0.09), but not between ciliary beat frequency and ciliary orientation angle (r = −0.08, p = 0.63). There was a significant correlation between NMCC time and age (r = 0.64; p < 0.001; Figure 2B) and central microtubular orientation angle (0.45, 0.04), but not between NMCC time and ciliary beat frequency (r = 0.21, p = 0.20) or percent of ciliary cross-sections exhibiting microtubular disarrangement (r = −0.56, p = 0.25), extra tubules (r = 0.48, p = 0.28), or single tubules (r = −0.28, p = 0.65).
Fig. 2. Scatterplots showing relationship between age and (A) ciliary beat frequency (n = 90), and (B) NMCC time (n = 43) in our cohort of subjects. Correlation lines were established with a simple linear regression method.
[More] [Minimize]The results of our study of 90 healthy or stable subjects, whose age ranged from 11 to 90 yr, showed that aging was associated with a decrease in ciliary beat frequency and increase in NMCC time, which negatively reflects the efficiency of NMCC. The orientation of the central microtubules of cilia, which reflected the direction of ciliary beat, did not change significantly with increasing age. There was no sex difference in ciliary beat frequency, NMCC time, or central microtubular orientation with aging. There was a significant increase in the occurrence of microtubular defects, including disarrangement of microtubules and the presence of extra microtubules or single microtubules, with aging. Ciliary beat frequency, NMCC time, and percent of ciliary cross-sections displaying single tubules were significantly different in subjects under and over 40 yr of age, respectively. Our original results therefore show that ciliary function declines with aging, and that this process is associated with an increasing occurrence of ultrastructural defects in respiratory epithelial cilia.
Only a few human studies of the effects of aging on ciliary function have been published, and the results, probably due to small sample size, have been inconclusive (6, 13-16). Using a similar photometric technique to ours, Yager and colleagues reported that ciliary beat frequency slowed with increasing age (13). However, their measurements were made with suspensions of ciliated respiratory cells obtained by bronchoscopic brushing and which had also been exposed to anesthetic agents that might have adversely affected ciliary beating. The study subjects were also predominantly respiratory disease patients and probably not representative of the general population (13). A similar study, of the ciliary beat frequency of nasal mucosa from 60 healthy subjects and 60 patients with middle ear diseases, showed no age-related changes in ciliary beat frequency (14). Radionuclear measurement of radioactive aerosol clearance showed that aging was associated with a decline in mucociliary clearance in 19 men (6), although this was not confirmed in a more recent study of 68 healthy subjects (15). Tracheal mucus velocity, measured by radiologic monitoring of the movement of bronchoscopically instilled radioopaque Teflon discs, was lower in seven elderly nonsmokers than in 10 young nonsmokers (mean age: 63 and 23 yr, respectively) (16). However, the study sample size was too small for the results to be clinically conclusive (16). The saccharine test, despite being an established bedside investigation for assessing NMCC, has not been used to evaluate the effects of aging on mucociliary clearance (19). Our results showed that 40.3% of subjects were not able to perform the saccharine test despite its overt simplicity.
The importance of respiratory ciliary function in the pathogenesis of pulmonary diseases is best illustrated by primary ciliary dyskinesia, a hereditary condition whereby ciliary beat is uncoordinated (dyskinetic), resulting in gross impairment of mucociliary clearance (20, 21). Clinical symptoms of primary ciliary dyskinesia include chronic or recurrent respiratory tract infections, bronchiectasis, and occasionally male sterility and situs inversus, which is typical of Kartagener's syndrome (22). The common ultrastructural ciliary defects, which have also been reported in apparently healthy subjects, include the complete absence or deficiency of outer or inner dynein arms, nexin links, radial spokes, the central sheath, inner microtubules, and even the entire axoneme (6, 21-27). More recently, ultrastructurally normal cilia showing normal beating have been found to be associated with a random central microtubular orientation. Such a random microtubular orientation has consequently been suggested to be a variant of primary ciliary dyskinesia leading to the development of bronchiectasis (3, 28). Although the percent of ciliary ultrastructural abnormalities associated with aging might initially appear low, we have recently shown that the presence of a ciliary ultrastructural abnormality as “infrequent” as in 2.3% of the total ciliary population could be associated with significant impairment of mucociliary clearance and development of severe bronchiectasis (18).
We used nasal rather than lower respiratory tract mucosa in our study, since the ciliary beat frequency of nasal mucosa correlates with that of tracheal mucosa (29). The use of nasal mucosa for assessment of ciliary function and ultrastructure is also firmly established (1, 2, 17), and obviates the need for more invasive procedures such as bronchoscopy. Because the structure and function of cilia are conserved among all species, the use of nasal cilia for assessment is both convenient and logical (23, 30). Despite their decline with aging, ciliary beat frequency and NMCC did not correlate with each other. This could have been due to the presence of other potential factors affecting mucociliary clearance. These include quantitative and qualitative changes in respiratory mucus with aging, age-related changes in anatomy, age-accumulated damage to the nasal mucosa from previous infection, trauma and exposure to external toxin(s), and other, unknown factors. These factors could also potentially either directly or indirectly lead to a decline in ciliary beat frequency and the occurrence of increased central microtubular disorientation with aging.
Pneumonia is the fourth leading cause of death in the world, and is the leading infectious cause of death in the elderly (7). Persons older than 65 yr of age account for half of all pneumonia cases (8). A number of physiologic and anatomic changes that impair respiratory function have been described in the elderly, although these should not predispose the elderly to respiratory infections. Although FEV1 and FVC decline with age independently of smoking or environmental and other factors (31), concurrent illnesses or deconditioning may also adversely influence pulmonary function (10). Reduction of small airway caliber, due to alterations in the cross-linkage of aging collagen and to decreased elasticity of extracellular matrix (11, 12), also occurs with aging. Aging-related diaphragmatic defects, resulting in the loss of diaphragmatic continuity (32), compound the adverse effects of loss in respiratory muscle power with aging (33). Cell-mediated immunity is largely unaffected by aging, and macrophage function, including adherence, chemotaxis, and phagocytosis, remains normal (9, 34). However, neutrophil chemotaxis and respiratory burst appear to be less effective with aging (35), and this might predispose the elderly to bacterial infection. In addition, an age-related decline in humoral immunity, largely due to a decrease in T-helper cells, occurs in the elderly (9). Our finding of impaired mucociliary clearance and ciliary function with aging, and of the increasing frequency of ciliary ultrastructural anomalies, might also help explain the frequent occurrence of respiratory infection in the elderly.
It is possible that the aforementioned defects culminate to an increased predisposition of the elderly to respiratory infection and a subsequent increase in morbidity and mortality. Further research should be done to elucidate the mechanism(s) underlying the slowing of ciliary beat and development of ciliary ultrastructural defects with aging.
Fig. 3. Transmission electron micrograph showing normal “9+2” structure of microtubules of two neighboring cilia whose central microtubules (each marked with a line) were in a similar orientation in a 45-yr-old subject. Original magnification: ×2,000.
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Fig. 4. Transmission electron micrograph of a cilium showing (A) disarrangement of microtubules in a 68-yr-old subject; (B) an extra microtubule (arrow) in an 85-yr-old subject; and (C) a single microtubule (arrow) in a 72-yr-old subject. Original magnification: ×3,000.
The authors are grateful to the subjects who donated specimens of their nasal mucosa, and to Dr. Ian Lauder of the Department of Statistics of the University of Hong Kong for expert statistical advice.
Supported by a Hong Kong RGC Competitive Grant (HKU7307/98M)
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