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Alignment of cilia in immotile-cilia syndrome
TISSUE & CELL 1986 18 (4) 521-529 @ 1986 Longman Group UK Ltd
M. C. HOLLEY*
ALIGNMENT SYNDROME
OF CILIA
Keywords:
and B. A. AFZELlUSt
IN IMMOTILE-CILIA
Cilia, ciliary alignment,
ciliary basal apparatus,
immotile-cilia
syndrome
ABSTRACT. Alignment of cilia in nasal epithelial cells from eight human subjects suffering from immotile-cilia syndrome was compared with that of cells from five control subjects. Individual cilia were assessed according to the orientation of their basal feet. The range of orientation of basal feet on a single cell varied from 26” to 261” and 54” to 275” in controls and patients respectively. Less than 10% of the cells from each group supported cilia that were aligned randomly. Alignment was worse in subjects with immotile-cilia syndrome but this could well have been due to secondary characteristics of the disease, such as common viral infection. Very accurate alignment of mucus-propelling cilia may be unnecessary. Measurements from control subjects and some invertebrates suggest that ranges of 140” are common and do not seriously impair mucus propulsion.
Introduction The metazoan cilium is anchored and orientated by the basal apparatus, a collection of structures that may include shiated rootlets, microtubules and microfilaments, which are attached to the basal body (Fawcett and Porter, 1954; Gibbons, 1961; Hard and Rieder, 1983; Holley, 3984,198.5; Pitelka, 1974; Reed et al., 1984). In genetic disorders of human cilia, collectively known as immotile-cilia syndrome (ICS), the axial orientation of cilia and their basal bodies, which determine the direction in which they beat, has in some cases been described as random (Afzelius, 1979, 1981; Afzelius et al., 1983; Nielsen et al., 1983; Pedersen, 1983; Pedersen et al., 1981; Rossman et al., 1981; Schneeberger et al., 1980; Veerman et al., 1980). This implies complete loss of the normal orientation mechanism. A study of ciliated epithelia from
*Department of Zoology, South Parks Road, Oxford OX1 3PS, England. Current address: Department of Physiology, The Medical School, University Walk, Bristol BS8 1TD. tDepanment of Ultrastructure Research, Biology E4, University of Stockholm, S-106 91 Stockholm, Sweden. To whom reprint requests should be addressed. Received
24 March 1986.
people suffering from ICS might, therefore, contribute further to our understanding of the problems and mechanisms of ciliary alignment. The human nasal mucosa is a pseudostratified, columnar epithelium composed of ciliated cells, goblet cells, basal cells, and intermediate cells resting upon a basement membrane (Jafek, 1983). Each ciliated cell bears 150-200 cilia, S-8pm long, resembling those described from tracheal epithelia (Rhodin, 1959; Rhodin and Dalhamn, 1956). The ciliary basal apparatus is composed of a basal body, derived from a centriole, from the side of which projects a single conical basal foot. A small striated rootlet projects downward from the lower end of the basal body. Transition fibres link the cilium base to the plasma membrane. Microtubules attached to the tip of the basal foot, and microfilaments associated less specifically with other parts of the apparatus, are integrated with the apical cytoskeleton thus uniting the basal bodies of all cilia from a single cell. The basal apparatus closely resembles that described in detail from human trachea (Gordon et al., 1980; Gordon, 1982), and from rhesus monkey oviduct (Anderson, 1972). Immotile-cilia syndrome, synonymous with the term ‘primary ciliary dyskinesia’ (Sleigh et al., 1981), is an autosomal recessive
522
disease affecting cilia and flagella (Palmblad etal., 1984). One in every two sufferers of the disease has a condition known as situs inversus, and, therefore, has the major symptoms of Kartagener’s syndrome, which is considered to be a subgroup of ICS (Afzehus, 1979; Sleigh et al., 1981; Rott, 1983; Sleigh, 1983). The disease is characterized by immotile or poorly motile cilia or flagella, caused by structural defects within the ciliary or flagellar axoneme; for example, partial or total loss of dynein arms, which drive the sliding microtubule mechanism (Fox ef al., 1981, 1983; Pedersen, 1983). Reports that patients with ICS have rancilia common domly orientated are throughout the literature, but only a few authors have quantified the phenomenon (e.g. Pedersen et al., 1981; Nielsen et al., 1983). Such assessments, however. were made by reference to the orientation of the central pair of axonemal microtubules, which in most animals are said to be orientated, at least at the base, perpendicular to the plane of the effective-stroke (Fawcett and Porter, 1954; Afzelius, 1961; Gibbons, 1961; Tamm and Horridge. 1970). Measurements of orientation are open to error because the central microtubule pair may rotate within the axoneme during ciliary movement (Omoto and Kung, 1979,198O) and cilia may twist during preparation for microscopy (Nielsen et al., 1983). It is better, therefore, to estimate ciliary alignment by measuring the angle at which the single basal foot projects from a cross-sectioned basal body (Sleigh, 1983); this has the added advantage that, not only does the basal foot indicate the plane of the ciliary effective-stroke, but also the direction. In most animals the basal foot projects from the side towards which the cihum moves during an effective-stroke (Fawcett and Porter, 1954; Gibbons, 1961; Hard and Rieder , 1983; Sleigh and Silvester , 1983; Holley, 1984), although this does not always apply to a cilium that can reverse the direction of its effective-stroke. This paper addresses the following questions using measurements of basal foot orientations from cells of control patients and patients suffering from ICS. (a) What is the accuracy of ciliary alignment in normal nasal ciliated epithelial cells? (b) Is alignment truly random in diseased cells, or at least significantly worse than in control cells? (c) Is poor
HOLLEY
alignment ICS?
AND AFZELIUS
likely to be a primary
feature
of
Methods Eight patients with full symptoms of ICS, including situs inversus, and five controls with no history of the disease, were selected. None of the patients were smokers. One control was a smoker, and another an ex-smoker; the latter also suffered from a common cold shortly before the biopsy was taken (see Table 1). Data were also collected from a person who did not have Kartagener’s syndrome or associated ultrastructural ciliary defects, but who had clinical symptoms of ICS and abnormally long cilia (Afzelius et al., 1985). A brush biopsy was obtained from the nasal mucosa of each person, fixed in glutaraldehyde. post-fixed in OsO,, dehydrated. and embedded in Epon. Thin sections were prepared on an LKB III ultramicrotome, and stained with uranyl acetate and lead citrate before being examined in a Jeol 100s transmission electron microscope. All sections were photographed at a magnification of x5000 and printed to a total magnification of X 10,000. The convex surface of each ciliated cell. and the difficulties of orientating the tissue for thin sectioning, meant that few measurable basal bodies could be sectioned simultaneously. Data were, therefore, collected from composites of serial sections, with measurements from at least ten basal feet from each cell. An arbitrary axis, approximately in line with the plane of the ciliary beat, was drawn for each sampled cell; since the cell apices were sectioned transversely this axis was parallel to the cell surface. Then a line was drawn each sectioned basal through body/basal foot complex, so that it passed through the tip of the basal foot and the centre of the transversely sectioned basal body (Fig. 1). The angle of each line was then measured relative to the arbitrary cell axis (Fig. 2). Then, for each cell sampled, the mean, range and variance of basal foot orientations were computed. Basal feet orientated near to 180” from the axis were measured as negative or positive values depending upon which produced the smallest variance (Fig. 2). The mean was only useful when two
ALIGNMENT OF CILIA IN IMMOTILE-CILIA
SYNDROME
523
then it should be equal across two 180” segments of the whole 360” range. The probability that an observed distribution differs from random can be computed as follows: x/n--0.5 z= 1(05x05)/n where x=number of basal feet in one 180 segment n=total number of basal feet measured z = has a normal probability distribution such that if z>1.96, P
or more neighbouring cells shared the same arbitrary axis and could be compared directly. To test for a random distribution of basal feet within a cell, the following procedure was employed: if the distribution is random
A summary of results for the five controls and eight patients is presented in Table 1. Values for both mean range and mean variance were highly sensitive to basal feet that were orientated well outside the general range for each cell: the frequency of such basal feet was clear from estimates of the percentage number that fell outside the 180” arc. This problem was controlled to some extent by computing range and variance from only 90% of the measurements for each cell, omitting those at the extremes. The statistical ‘weight’ of extreme values is decreased considerably by simply counting the number of
Fig. 2. (a) Diagram of a cross-sectioned cell apex (which normally bears 150-200 cilia). The angle measurements from 18 basal feet were taken from a cell from patient 6 (Table 1). Each, basal foot was measured relative to an arbitrary axis as shown. Badly aligned basal feet were given positive or negative values depending upon which gave the smallest variance, e.g. arrowed basal foot= - 137”, not 223”. (b) A pie chart, derived from (a), gave a better idea of alignment. In this ceU n=18, n-10%=16, range=129”, variance=980, percentage basal feet outside 60” and 180” arcs respectively are 33 and 11%. The distribution is not random. The arrow corresponds to the basal foot marked by an arrow in (a).
524
HOLLEY AND AFZELIUS
Table 1. No. cells
Controls 1 2* 3 4 St Patients 1 2 3 4 5 6 7 8 ‘A’
Mean (min-max) range
Mean (minmax) variance
% outside 60” arc
% outside 180” arc
No. cells random
162 44 118 192 93
7.5”(26122) 82” (41-116) 84” (28-146) 141” (45-261) 163” (51-254)
436 (461457) 671 (144-1190) 689 (62-1962) 1649 (228-7364) 2867 (346-5170)
13.0 15.9 19.5 27.6 45.2
1.2 4.5 1.7 8.3 10.8
2
80” (60-93) 113” (83-154) 143” (64-242) 154” (59-237) 1540 (87-211 j 182” (65-275) 183” (13F246) 200” (54-263)
533 (331-773) 1516 (660-2642) 2084 (372-4747) 2152 (462-4422 j 2228 (773-3919) 2903 (445-4886) 3470 (12964872) 3503 (437-5883)
18.6 35.0 31.4 35.0 41.3 43.0 53-5 44.4
1.7 2.5 11.4 10.6 6.3 14.0 13.1 15.9
0 0
8
59 40 35 160 63 86 99 126
48
1176
49” (14-84)
151 (17-499)
7-2
1.4
0
4 9 10 6
5
3 3 10 4 5
No. basal feet
0 0
Measurements from five controls and eight patients. *=smoker. t=ex-smoker with a common cold before biopsy. Patients l-8 had Kartagener’s syndrome: patient ‘A’ possessed abnormally long cilia and the basal bodies were often arranged in rows, but there were no other obvious defects. At least ten basal feet were measured from each cell sampled. Mean range and mean variance were computed from 90% of the recorded values in each cell, and are presented with minimum and maximum values for each person. The test for random alignment is described under ‘Methods’
measurements
that fall outside
a given range;
proportions can then be employed for direct comparison. Extensive variability of basal foot orientation was recorded within cells, between cells, and between people, both in control and patient groups. The mean ranges, mean variances, and percentage number of basal feet outside a 60“ arc, ranked in ascending order, all produced the same value for ‘U’ in the Mann-Whitney U-test (L/=9, ni=5, n2=8; P=OJl64). This gave a strong indication that there was a significant difference between the patient and control groups, although it was obviously not clear-cut. if control five, who had a common cold before the biopsy, was omitted, then the difference was more significant; with respect to mean variance and mean range, U=4, P=O.O24, and with respect to the percentage of basal feet lying outside a 60” arc U=2, P=O.O08. There was virtually no overlap between minimum ranges and variances from cells in the control and patient groups, but this was certainly not true of the maximum values.
The number of cells with a distribution insignificantly different from random was less than 10% in both groups; three from 36, and four from 45 in control and patient groups respectively. Where the arbitrary axis given to a cell could be extended to a neighbouring cell that contained at least ten measurable basal feet, the differences between the means was calculated (Table 2). There was no strong indication that cilia were misaligned between cells, with the possible exception of patient 7. It must be remembered, however, that mean values are much less accurate from small samples that have large variances. Patient ‘A’, who had clinical symptoms of ICS resulting from abnormally long cilia, displayed exceptionally well-orientated basal feet, with a mean range of 49”, a mean variance of 151, and with 1.2 and 6.3% of the measurements falling outside 180” and 60” arcs respectively. The basal bodies formed closely packed rows in which the basal feet were apparently in contact with neighbouring basal bodies (Fig. 3). Consequently many basal feet in a particular row had almost iden-
ALIGNMENT
OF CILIA IN IMMOTILE-CILIA
SYNDROME
mucus-propelling cilia in B. lanceolatum and C . parasitica produce ranges of
from
Table 2 Difference between means of neighbouring cells Control 4 5
10.9 23.8”
Patient 4
15.0” 0.8” 56.8” 28.6 32.6 26.2” 72.9” 123.0” 8.1”
5 6 7
8 ‘A’
525
11.3k6.7” (27)
Where fortuitous sections passed through two neighbouring cells with at least ten basal feet in each, they were given the same arbitrary axis and
their mean angles were compared. For patient ‘A’ the figures are meanfstandard deviation (no. cell pairs).
tical orientations. Mean values between cells indicated differences ranging from 0” to 58” (Table 2). Discussion
An ideal ciliated epithelium, composed of cilia aligned exactly in parallel, would require accurate alignment not only of cilia within each cell but also of all cells within that epithelium (see Sleigh, 1983). Such precision is but how much unnecessary, clearly variability can be tolerated before fluid propulsion is impaired? The answer will to some extent depend upon the function of the cilia. Cilia that propel water are generally better coordinated than those that propel mucus (Sleigh, 1976), and there is some evidence that their alignment is more critical (Holley, 1984). Measurements from basal feet of such cilia in the protochordate Branchiostoma lanceolatum and the coelenterate Calliactisparasitica produce ranges of 10°-40” with variances of 5-65; similar values may be expected from measurements of central microtubule pairs in other types of waterpropelling cilia, for example in mussel gill (Fig. 39, Gibbons, 1961). Measurements
53”140” with variances of 100-900; values that clearly overlap with those recorded in this study. A mucus sheet is expected to average the forces produced by the cilia that act upon it, thus restraining forces produced by individual disorientated cilia; if cilia on a particular cell were randomly orientated across a 180” arc, then they should propel a mucus sheet along a line bisecting that arc. This is unlikely to apply to cilia that propel water because water has a much lower viscosity and consequently exerts less force upon them. In this study the percentage number of basal feet that lie outside a 60” arc corresponds well to similar measurements using central microtubule pairs, in which values of l-26 and l&45 % were recorded from control and patient groups respectively (Pedersen et al., 1981; the measurement, ‘percentage number differing by more than 30” to the general axis’, is equivalent to a 60” range). Further data recording mean standard deviations for control and patient groups, again based on the central microtuble pair, produced values equivalent to variances of about 80-800 and 230-2025 respectively (Nielsen et al., 1983). The measurements from both sources probably underestimate the variance because they relate only to the plane of the ciliary effective-strokes, and not to their direction; 0” is thus indistinguishable from 180”. Nevertheless it appears that the alignment of mucus-propelling cilia is normally poor, but that they are able to propel mucus effectively even if their planes of beat, as assessed by electron microscopy, diverge by as much as 140”. Very few cells, either from the controls or the patients, possessed cilia aligned at random, although alignment was significantly worse in the patient group. The large variability between cells in each patient indicates that alignment was relatively good in some cells, and hence that the alignment mechanism was intact, but that in others alignment had been disrupted. Poor alignment is, therefore, unlikely to be a primary feature of ICS, at least in the eight patients studied here, and may instead be caused by secondary effects such as viral infection, or even mechanical damage caused by coughing; both clinical symptoms of ICS (Afzelius,
ALIGNMENT
OF CILIA
IN IMMOTILE-CILIA
1979). Poor alignment of cilia has been correlated with respiratory tract infections (Holwell et al., 1980; Lungarella et al., 1983), and asthma (Laitinen et al., 1985). Furthermore, disorientated ciliary beat patterns have been described in the tracheobronchial tree of bronchitic rats (Iravani and Van As, 1972). We may conclude that alignment is likely to be worse among patients suffering from ICS because of their increased rate of infection, and that poor alignment in control 5 was a result of infection immediately before sampling. It might, therefore, be profitable to test alignment of cilia in ciliated cells from people with and without common infections. Some variation in ciliary alignment in different cells within the same epithelium might arise through sampling cells which are at different stages of their life-cycle. In nasal epithelia from embryonic mice, development of rootlets, basal feet and cilia precedes alignment (Frisch and Farbman, 1968), and in quail oviduct (Boisvieux-Ulrich et al., 1985) and rat lung (Sorokin, 1968) there is evidence that cilia are not aligned until they are mature and start beating. Sections through developing cells in normal epithelia might, therefore, show cilia that are poorly (randomly?) aligned. Furthermore, alignment might deteriorate with cell age, or at least deteriorate rapidly at cell death, thus producing a similar result, but in the absence of a method for measuring cell age this is difficult to establish. Afzelius (1981) suggested that basal bodies, unorientated when they migrate to the cell surface during development, are aligned by the act of beating, via the same hydromechanical forces that lead to metachronal coordination (see Machemer, 1975); if cilia are defective and unable to beat properly, as they often are in ICS, then alignment would not occur. This idea is difficult to test. In quail oviduct alignment is closely related, not only to the initiation of ciliary beating, but also to differentiation of the cortical
Fig. 3. Cross-sectioned Alignment was abnormally
cells from patient good. x22,5300.
527
SYNDROME
cytoskeleton, particularly of the microtubules and microfilaments associated with the basal feet (Boisvieux-Ulrich et al., 1985). If beating does help to align cilia then it may be a transient phenomenon with the basal bodies soon locked into place by the cortical cytoskeleton; it might even be the stimulus for cytoskeletal differentiation. Cilia on microsurgically reversed segments of rabbit oviduct maintain their original beat direction, against that of neighbouring cilia, for at least 13 months after the operation (Eddy et al., 1982), and cilia on fragments of cortex reversed through 180” in the protozoan Paramecium aurelia likewise maintain their original beat direction against that of neighbouring cilia, even through successive generations (Beisson and Sonneborn, 1965; Tamm et al., 1975). Assuming that the basal apparatus/cilium complex behaves as a unit, we may conclude that disruption of ciliary alignment involves general disruption of the cytoskeleton, which may affect alignment between neighbouring cells as well as between cilia within a cell. Many of the basal bodies in patient ‘A’ were arranged in rows, and alignment of the basal feet was particularly good, but the effects of this in terms of the function of the cilia is not clear. The arrangement, however, is unusual in human ciliated cells and we can assume that cytoskeletal elements normally not only help to anchor cilia with a suitable orientation, but also help to regulate their spatial distribution (see Reed et al., 1984). This process is not yet properly understood in any cell. Throughout this discussion we have assumed that the basal foot has a constant position relative to the ciliary axoneme, even in the patient group. This seems to be reasonable on the grounds that throughout the animal kingdom it has a predictable orientation relative to the ciliary effectivestroke, and that variation in basal foot alignment, as discussed above, is similar to that of
‘A’. The basal bodies
were arranged
in rows.
HOLLEY
the central microtubule pairs. The assumption could be tested by serial sectioning, particularly of cells in which alignment is very poor; until then, the possibility that the basal foot is mislocated on the basal body should not be ignored. Basal bodies sometimes support more than one basal foot (see Lungarella et al., 1985), thus illustrating the potential for a basal foot to be attached to a basal body at more than one site. In this report 1.1 and 2.6% of basal bodies, in controls and patients respectively, supported two&Sal feet. We may conclude that nasal cilia, in common with other mucus-propelling cilia, are not very accurately aligned. Alignment in patients suffering from ICS is rarely random, although generally less accurate than in controls, and it cannot reasonably be considered a primary feature of the disease. Poor align-
AND AFZELIlJS
ment is usually associated with a wide variety of other cellular and ciliary defects so it would not be easy to assess the effects of disorientation alone on mucus-propelling efficiency. There is little doubt, however, that poor alignment can only contribute to the problems of mucus clearance.
Acknowledgements
We thank those people who volunteered to undergo a nasal biopsy, Mrs Ulla Afzelius for patiently preparing sections, Drs R. W. Hiorns and Sophie Matthiesen for statistical advice, and Professor M. A. Sleigh for reading the manuscript. M.C.H. was supported by a Royal Society European Exchange Programme Fellowship.
References Afzelius, B. A. 1961. The fine structure of the cilia from ctenophore swimming plates. J. biophys. biochem. Cytol., 9, 38S394. Afzelius, B. A. 1979. The immotile-cilia syndrome and other ciliary diseases. Int. Rev. exp. Path., 19, 1-43. Afzelius, B. A. 1981. Genetic disorders of cilia. In International Cell Biology 1980-1981, pp.440-447. Springer-Verlag. Berlin and Heidelberg. Afzeiius, B. A., Camner, P. and Mossberg, B. 1983. Acquired ciliary defects compared to those seen in the immotilecilia syndrome. Eur. .I. Respir. Dis., 64 (Suppl. 127). l--1. Afzelius, B. A., Gargani, G. and Romano, C. 1985. Abnormal length of cilia as a possible cause of defective mucociliary clearance. Eur. 1. Respir. Dir. 66, 17f180. Anderson, R. G. W. 1972. The three-dimensional structure of the basal body from the rhesus monkey oviduct. J. Cell Biol., 54, 246265. Beisson, J. and Sonneborn, T. M. 1965. Cytoplasmic inheritance of the organisation of the cell cortex in Paramecium aurelia. Proc. mtn. Acad. Sci. 53, 275-282. Boisvieux-Ulrich, E., Laine, M.-C. and Sandoz, D. 1985. The orientation of ciliary basal bodies m quail oviduct is related to the ciliary beating cycle commencement. Biol. Cell. 55, 147-150. Eddy, C. A., Archer, D. R. and Pauerstein, C. J. 1982. Failure of cilia to reprogram following segmental ampullary reversal of rabbit oviduct. Experientia, 38, 104-105. Fawcett, D. W. and Porter, K. R. 1954. A study of the fine structure of ciliated epithelia. 1. Morph.. 94, 221-281. Fox, B., Bull, T. B., Makey, A. R. and Rawbone, R. 1981. The significance of ultrastructural abnormalities of human cilia. Chest, 80, 796799. Fox, B., Bull, T. B. and Oliver, T. N. 1983. The distribution and assessment of electron-microscopic abnormalities of human cilia. Eur. J. Resprr. Dis., 64, (Suppl.127). 11-18. Frisch, D. and Farbman, A. 1. 1968. Development of order during ciliogenesis. Amt. Rec.. 162,221-232. Gibbons, 1. R. 1961. The relationship between the fine structure and the direction of beat in gill cilia of a lamellibranch mollusc. J. biophys. biochem. Cytol., 11, 179-205. Gordon, R. E. 1982. Three-dimensional organisation of microtubules and microfilaments of the basal body apparatus of ciliated respiratory epithelium. Cell Motility, 4, 385-391. Gordon, R. E., Lane, B. P. and Miller, F. 1980. Identification of contractile protems in basal bodies of cihated tracheal epithelial cells. J. Histochem. Cytochem., 28, 1189-1197. Hard, R. and Rieder, C. L. 1983. Mucociliary transport in newt lungs: the ultrastructure of the ciliary apparatus in isolated epithelial sheets and in functional triton-extracted models. Tissue & Cell, 15, 227-243. HoUey, M. C. 1984. The ciliary basal apparatus is adapted to the structure and mechanics of the ciliated epithelium. Tissue & Cell, 16,287-310.
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SYNDROME
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Halley, M. C. 1985. Adaptation of a ciliary basal apparatus to cell shape changes in a contractile epithelium. Tissue & Cell, 17, 321-334. Holwell, J. T., Schochet, S. S. and Golman, A. S. 1980. Ultrastructural defects of respiratory tract cilia associated with chronic infections. Arch. Parhol. Lab. Med., 104, 52-55. Iravani, J. and Van As, A. 1972. Mucus transport in the tracheobronchial tree of normal and bronchitic rats. J. Parhol., 106, 81-93. Jafek, B. W. 1983. Ultrastructure of human nasal mucosa. Laryngoscope, 93, 15761599. Laitinen, L. A., Heino, M., Laitinen, A., Kava, T. and Haahtela, T. 1985. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am. Rev. Respir. Db., 131, 59!%%. Lungarella, G., Fonzi, L. and Ermini, G. 1983. Abnormalities of bronchial cilia in patients with chronic bronchitis. Lung, 161, 147-156. Lungarella, G., De Santi, M. M., Palatresi, R. and Tosi, P. 1985. Ultrastructural observations on basal apparatus of respiratory cilia in immotile-cilia syndrome. Eur. J. Respir. Db., 66, 165-172. Machemer, H. 1975. Mechanical conditions of flagellar and ciliary metachronism. In SwimmingandFlyingin Nature (eds T. Y. Wu, C. J. Brokaw and C. Brennen), Vol. 1, pp. 211-221. Plenum Press, New York. Nielsen, M. H., Pedersen, M., Christensen, B. and Mygind, N. 1983. Blind quantitative electron microscopy of cilia from patients with primary ciliary dyskinesia and from normal subjects. Eur. I. Respir. DL., 64 (Suppl. 127). 1930. Omoto, C. K. and Kung, C. 1979. The pair of central microtubules rotates during ciliary beat in Paramecium. Nature, 279, 532-534. Omoto, C. K. and Kung, C. 1980. Rotation and twist of the central-pair microtubules in the cilia of Paramecium. 1. Cell Eiol., 87, 3346. Palmblad, J., Mossberg, B. and Afzelius, B. A. 1984. Ultrastructural, cellular, and clinical features of the immotile-cilia syndrome. A. Rev. Med., 35,481-492. Pedersen, M., Morkassel, E., Nielsen, M. H. and Mygind, N. 1981. Kartagener’s syndrome. Preliminary report on cilia structure, function, and upper airway symptoms. Chesr, 80, 858-860. Pedersen, M. 1983. Specific types of abnormal ciliary motility in Kartagener’s syndrome and analgous respiratory disorders. Eur. J. Respir. Dis., 64 (Suppl. 127). 78-90. Pitelka, D. R. 1974. Basal bodies and root structures. In Cilia and Flagella (ed. M. A. Sleigh), pp. 437-469. Academic Press, London. Reed, W., Avolio, J. A. and Satir, P. 1984. The cytoskeleton of the apical border of the lateral cells of freshwater mussel gill: a structural integration of microtubules and actin filament-based organelles. 1. Cell Sci., 68, l-33. Rhodin, J. (1959) Ultrastructure of the tracheal ciliated mucosa in rat and man. Ann. Otol. Rhin. Laryng., 68,964. Rhodin, J. and Dalhamn, T. 1956. Electron microscopy of the tracheal ciliated mucosa in rat. 2. Zellforsch. mikrosk. Anat., 44, 345-412. Rossman, C. M., Forrest, J. B., Lee, R. M. K. W., Newhouse, A. F. and Newhouse, M. T. 1981. The dyskinetic cilia syndrome. Abnormal ciliary motility in association with abnormal ciliary ultrastructure. Chesr, 80, 8-865. Rott, H. D. 1983. Genetics of Kartagener’s syndrome. Eur. 1. Respir. Db., 64 (Suppl. 127), 1-4. Schneeberger, E. E., McCormack, J., Issenberg, H. J., Schuster, S. R. and Gerald, P. S. 1980. Heterogeneity of ciliary morphology in the imrnotile-cilia syndrome in man. J. Ulwasrrucr. Res., 73, 34-43. Siegel, S. (1956) Non-parametric .%&tics for the Eehavioural Sciences. McGraw-Hill, Kogakusha. Sleigh, M. A. (1976) Fluid propulsion by cilia and the physiology of ciliary systems. In Perspectives in Experimenral Biology (ed. P. Spencer-Davies), pp. 125-134. Pergamon Press, Oxford. Sleigh, M. A. 1983. Kartagener’s syndrome, ciliary defects and ciliary function. Eur. J. Respir. Dis., 64, (Suppl. 127), 157-161. Sleigh, M. A. and Silvester, N. R. 1983. Anchorage functions of the basal apparatus of cilia. J. submicrosc. Cyrol., 15, 101-104. Sleigh, M. A. and others. 1981. Primary ciliary dyskinesia (letter). Lancer, 2, 476. Sorokin, S. P. 1968. Reconstructions of ccntriole formation and ciliogenesis in mammalian lungs. J. Cell Sci. 3,207-230. Tarmn, S. L. and Horridge, G. A. 1970. The relationship between the orientation of central fibrils and the direction of beat in cilia of Opalina. Proc. R. Sot. Lond., Ser. B, 175, 219233. Tamm, S. L., Sonneborn, T. M. and Dippell, R. V. 1975. The role of cortical orientation in the control of the direction of ciliary beat in Paramecium. 1. Cell Biol., 64, 98-112. Veerman, A. J. P., Van Delden, L., Feenstra, L. and Leene, W. 1980. The immotile-cilia syndrome: phase contrast light microscopy, scanning and transmission electron microscoy. Pediatrics, 65, 698-702.
35
M. C. HOLLEY*
ALIGNMENT SYNDROME
OF CILIA
Keywords:
and B. A. AFZELlUSt
IN IMMOTILE-CILIA
Cilia, ciliary alignment,
ciliary basal apparatus,
immotile-cilia
syndrome
ABSTRACT. Alignment of cilia in nasal epithelial cells from eight human subjects suffering from immotile-cilia syndrome was compared with that of cells from five control subjects. Individual cilia were assessed according to the orientation of their basal feet. The range of orientation of basal feet on a single cell varied from 26” to 261” and 54” to 275” in controls and patients respectively. Less than 10% of the cells from each group supported cilia that were aligned randomly. Alignment was worse in subjects with immotile-cilia syndrome but this could well have been due to secondary characteristics of the disease, such as common viral infection. Very accurate alignment of mucus-propelling cilia may be unnecessary. Measurements from control subjects and some invertebrates suggest that ranges of 140” are common and do not seriously impair mucus propulsion.
Introduction The metazoan cilium is anchored and orientated by the basal apparatus, a collection of structures that may include shiated rootlets, microtubules and microfilaments, which are attached to the basal body (Fawcett and Porter, 1954; Gibbons, 1961; Hard and Rieder, 1983; Holley, 3984,198.5; Pitelka, 1974; Reed et al., 1984). In genetic disorders of human cilia, collectively known as immotile-cilia syndrome (ICS), the axial orientation of cilia and their basal bodies, which determine the direction in which they beat, has in some cases been described as random (Afzelius, 1979, 1981; Afzelius et al., 1983; Nielsen et al., 1983; Pedersen, 1983; Pedersen et al., 1981; Rossman et al., 1981; Schneeberger et al., 1980; Veerman et al., 1980). This implies complete loss of the normal orientation mechanism. A study of ciliated epithelia from
*Department of Zoology, South Parks Road, Oxford OX1 3PS, England. Current address: Department of Physiology, The Medical School, University Walk, Bristol BS8 1TD. tDepanment of Ultrastructure Research, Biology E4, University of Stockholm, S-106 91 Stockholm, Sweden. To whom reprint requests should be addressed. Received
24 March 1986.
people suffering from ICS might, therefore, contribute further to our understanding of the problems and mechanisms of ciliary alignment. The human nasal mucosa is a pseudostratified, columnar epithelium composed of ciliated cells, goblet cells, basal cells, and intermediate cells resting upon a basement membrane (Jafek, 1983). Each ciliated cell bears 150-200 cilia, S-8pm long, resembling those described from tracheal epithelia (Rhodin, 1959; Rhodin and Dalhamn, 1956). The ciliary basal apparatus is composed of a basal body, derived from a centriole, from the side of which projects a single conical basal foot. A small striated rootlet projects downward from the lower end of the basal body. Transition fibres link the cilium base to the plasma membrane. Microtubules attached to the tip of the basal foot, and microfilaments associated less specifically with other parts of the apparatus, are integrated with the apical cytoskeleton thus uniting the basal bodies of all cilia from a single cell. The basal apparatus closely resembles that described in detail from human trachea (Gordon et al., 1980; Gordon, 1982), and from rhesus monkey oviduct (Anderson, 1972). Immotile-cilia syndrome, synonymous with the term ‘primary ciliary dyskinesia’ (Sleigh et al., 1981), is an autosomal recessive
522
disease affecting cilia and flagella (Palmblad etal., 1984). One in every two sufferers of the disease has a condition known as situs inversus, and, therefore, has the major symptoms of Kartagener’s syndrome, which is considered to be a subgroup of ICS (Afzehus, 1979; Sleigh et al., 1981; Rott, 1983; Sleigh, 1983). The disease is characterized by immotile or poorly motile cilia or flagella, caused by structural defects within the ciliary or flagellar axoneme; for example, partial or total loss of dynein arms, which drive the sliding microtubule mechanism (Fox ef al., 1981, 1983; Pedersen, 1983). Reports that patients with ICS have rancilia common domly orientated are throughout the literature, but only a few authors have quantified the phenomenon (e.g. Pedersen et al., 1981; Nielsen et al., 1983). Such assessments, however. were made by reference to the orientation of the central pair of axonemal microtubules, which in most animals are said to be orientated, at least at the base, perpendicular to the plane of the effective-stroke (Fawcett and Porter, 1954; Afzelius, 1961; Gibbons, 1961; Tamm and Horridge. 1970). Measurements of orientation are open to error because the central microtubule pair may rotate within the axoneme during ciliary movement (Omoto and Kung, 1979,198O) and cilia may twist during preparation for microscopy (Nielsen et al., 1983). It is better, therefore, to estimate ciliary alignment by measuring the angle at which the single basal foot projects from a cross-sectioned basal body (Sleigh, 1983); this has the added advantage that, not only does the basal foot indicate the plane of the ciliary effective-stroke, but also the direction. In most animals the basal foot projects from the side towards which the cihum moves during an effective-stroke (Fawcett and Porter, 1954; Gibbons, 1961; Hard and Rieder , 1983; Sleigh and Silvester , 1983; Holley, 1984), although this does not always apply to a cilium that can reverse the direction of its effective-stroke. This paper addresses the following questions using measurements of basal foot orientations from cells of control patients and patients suffering from ICS. (a) What is the accuracy of ciliary alignment in normal nasal ciliated epithelial cells? (b) Is alignment truly random in diseased cells, or at least significantly worse than in control cells? (c) Is poor
HOLLEY
alignment ICS?
AND AFZELIUS
likely to be a primary
feature
of
Methods Eight patients with full symptoms of ICS, including situs inversus, and five controls with no history of the disease, were selected. None of the patients were smokers. One control was a smoker, and another an ex-smoker; the latter also suffered from a common cold shortly before the biopsy was taken (see Table 1). Data were also collected from a person who did not have Kartagener’s syndrome or associated ultrastructural ciliary defects, but who had clinical symptoms of ICS and abnormally long cilia (Afzelius et al., 1985). A brush biopsy was obtained from the nasal mucosa of each person, fixed in glutaraldehyde. post-fixed in OsO,, dehydrated. and embedded in Epon. Thin sections were prepared on an LKB III ultramicrotome, and stained with uranyl acetate and lead citrate before being examined in a Jeol 100s transmission electron microscope. All sections were photographed at a magnification of x5000 and printed to a total magnification of X 10,000. The convex surface of each ciliated cell. and the difficulties of orientating the tissue for thin sectioning, meant that few measurable basal bodies could be sectioned simultaneously. Data were, therefore, collected from composites of serial sections, with measurements from at least ten basal feet from each cell. An arbitrary axis, approximately in line with the plane of the ciliary beat, was drawn for each sampled cell; since the cell apices were sectioned transversely this axis was parallel to the cell surface. Then a line was drawn each sectioned basal through body/basal foot complex, so that it passed through the tip of the basal foot and the centre of the transversely sectioned basal body (Fig. 1). The angle of each line was then measured relative to the arbitrary cell axis (Fig. 2). Then, for each cell sampled, the mean, range and variance of basal foot orientations were computed. Basal feet orientated near to 180” from the axis were measured as negative or positive values depending upon which produced the smallest variance (Fig. 2). The mean was only useful when two
ALIGNMENT OF CILIA IN IMMOTILE-CILIA
SYNDROME
523
then it should be equal across two 180” segments of the whole 360” range. The probability that an observed distribution differs from random can be computed as follows: x/n--0.5 z= 1(05x05)/n where x=number of basal feet in one 180 segment n=total number of basal feet measured z = has a normal probability distribution such that if z>1.96, P
or more neighbouring cells shared the same arbitrary axis and could be compared directly. To test for a random distribution of basal feet within a cell, the following procedure was employed: if the distribution is random
A summary of results for the five controls and eight patients is presented in Table 1. Values for both mean range and mean variance were highly sensitive to basal feet that were orientated well outside the general range for each cell: the frequency of such basal feet was clear from estimates of the percentage number that fell outside the 180” arc. This problem was controlled to some extent by computing range and variance from only 90% of the measurements for each cell, omitting those at the extremes. The statistical ‘weight’ of extreme values is decreased considerably by simply counting the number of
Fig. 2. (a) Diagram of a cross-sectioned cell apex (which normally bears 150-200 cilia). The angle measurements from 18 basal feet were taken from a cell from patient 6 (Table 1). Each, basal foot was measured relative to an arbitrary axis as shown. Badly aligned basal feet were given positive or negative values depending upon which gave the smallest variance, e.g. arrowed basal foot= - 137”, not 223”. (b) A pie chart, derived from (a), gave a better idea of alignment. In this ceU n=18, n-10%=16, range=129”, variance=980, percentage basal feet outside 60” and 180” arcs respectively are 33 and 11%. The distribution is not random. The arrow corresponds to the basal foot marked by an arrow in (a).
524
HOLLEY AND AFZELIUS
Table 1. No. cells
Controls 1 2* 3 4 St Patients 1 2 3 4 5 6 7 8 ‘A’
Mean (min-max) range
Mean (minmax) variance
% outside 60” arc
% outside 180” arc
No. cells random
162 44 118 192 93
7.5”(26122) 82” (41-116) 84” (28-146) 141” (45-261) 163” (51-254)
436 (461457) 671 (144-1190) 689 (62-1962) 1649 (228-7364) 2867 (346-5170)
13.0 15.9 19.5 27.6 45.2
1.2 4.5 1.7 8.3 10.8
2
80” (60-93) 113” (83-154) 143” (64-242) 154” (59-237) 1540 (87-211 j 182” (65-275) 183” (13F246) 200” (54-263)
533 (331-773) 1516 (660-2642) 2084 (372-4747) 2152 (462-4422 j 2228 (773-3919) 2903 (445-4886) 3470 (12964872) 3503 (437-5883)
18.6 35.0 31.4 35.0 41.3 43.0 53-5 44.4
1.7 2.5 11.4 10.6 6.3 14.0 13.1 15.9
0 0
8
59 40 35 160 63 86 99 126
48
1176
49” (14-84)
151 (17-499)
7-2
1.4
0
4 9 10 6
5
3 3 10 4 5
No. basal feet
0 0
Measurements from five controls and eight patients. *=smoker. t=ex-smoker with a common cold before biopsy. Patients l-8 had Kartagener’s syndrome: patient ‘A’ possessed abnormally long cilia and the basal bodies were often arranged in rows, but there were no other obvious defects. At least ten basal feet were measured from each cell sampled. Mean range and mean variance were computed from 90% of the recorded values in each cell, and are presented with minimum and maximum values for each person. The test for random alignment is described under ‘Methods’
measurements
that fall outside
a given range;
proportions can then be employed for direct comparison. Extensive variability of basal foot orientation was recorded within cells, between cells, and between people, both in control and patient groups. The mean ranges, mean variances, and percentage number of basal feet outside a 60“ arc, ranked in ascending order, all produced the same value for ‘U’ in the Mann-Whitney U-test (L/=9, ni=5, n2=8; P=OJl64). This gave a strong indication that there was a significant difference between the patient and control groups, although it was obviously not clear-cut. if control five, who had a common cold before the biopsy, was omitted, then the difference was more significant; with respect to mean variance and mean range, U=4, P=O.O24, and with respect to the percentage of basal feet lying outside a 60” arc U=2, P=O.O08. There was virtually no overlap between minimum ranges and variances from cells in the control and patient groups, but this was certainly not true of the maximum values.
The number of cells with a distribution insignificantly different from random was less than 10% in both groups; three from 36, and four from 45 in control and patient groups respectively. Where the arbitrary axis given to a cell could be extended to a neighbouring cell that contained at least ten measurable basal feet, the differences between the means was calculated (Table 2). There was no strong indication that cilia were misaligned between cells, with the possible exception of patient 7. It must be remembered, however, that mean values are much less accurate from small samples that have large variances. Patient ‘A’, who had clinical symptoms of ICS resulting from abnormally long cilia, displayed exceptionally well-orientated basal feet, with a mean range of 49”, a mean variance of 151, and with 1.2 and 6.3% of the measurements falling outside 180” and 60” arcs respectively. The basal bodies formed closely packed rows in which the basal feet were apparently in contact with neighbouring basal bodies (Fig. 3). Consequently many basal feet in a particular row had almost iden-
ALIGNMENT
OF CILIA IN IMMOTILE-CILIA
SYNDROME
mucus-propelling cilia in B. lanceolatum and C . parasitica produce ranges of
from
Table 2 Difference between means of neighbouring cells Control 4 5
10.9 23.8”
Patient 4
15.0” 0.8” 56.8” 28.6 32.6 26.2” 72.9” 123.0” 8.1”
5 6 7
8 ‘A’
525
11.3k6.7” (27)
Where fortuitous sections passed through two neighbouring cells with at least ten basal feet in each, they were given the same arbitrary axis and
their mean angles were compared. For patient ‘A’ the figures are meanfstandard deviation (no. cell pairs).
tical orientations. Mean values between cells indicated differences ranging from 0” to 58” (Table 2). Discussion
An ideal ciliated epithelium, composed of cilia aligned exactly in parallel, would require accurate alignment not only of cilia within each cell but also of all cells within that epithelium (see Sleigh, 1983). Such precision is but how much unnecessary, clearly variability can be tolerated before fluid propulsion is impaired? The answer will to some extent depend upon the function of the cilia. Cilia that propel water are generally better coordinated than those that propel mucus (Sleigh, 1976), and there is some evidence that their alignment is more critical (Holley, 1984). Measurements from basal feet of such cilia in the protochordate Branchiostoma lanceolatum and the coelenterate Calliactisparasitica produce ranges of 10°-40” with variances of 5-65; similar values may be expected from measurements of central microtubule pairs in other types of waterpropelling cilia, for example in mussel gill (Fig. 39, Gibbons, 1961). Measurements
53”140” with variances of 100-900; values that clearly overlap with those recorded in this study. A mucus sheet is expected to average the forces produced by the cilia that act upon it, thus restraining forces produced by individual disorientated cilia; if cilia on a particular cell were randomly orientated across a 180” arc, then they should propel a mucus sheet along a line bisecting that arc. This is unlikely to apply to cilia that propel water because water has a much lower viscosity and consequently exerts less force upon them. In this study the percentage number of basal feet that lie outside a 60” arc corresponds well to similar measurements using central microtubule pairs, in which values of l-26 and l&45 % were recorded from control and patient groups respectively (Pedersen et al., 1981; the measurement, ‘percentage number differing by more than 30” to the general axis’, is equivalent to a 60” range). Further data recording mean standard deviations for control and patient groups, again based on the central microtuble pair, produced values equivalent to variances of about 80-800 and 230-2025 respectively (Nielsen et al., 1983). The measurements from both sources probably underestimate the variance because they relate only to the plane of the ciliary effective-strokes, and not to their direction; 0” is thus indistinguishable from 180”. Nevertheless it appears that the alignment of mucus-propelling cilia is normally poor, but that they are able to propel mucus effectively even if their planes of beat, as assessed by electron microscopy, diverge by as much as 140”. Very few cells, either from the controls or the patients, possessed cilia aligned at random, although alignment was significantly worse in the patient group. The large variability between cells in each patient indicates that alignment was relatively good in some cells, and hence that the alignment mechanism was intact, but that in others alignment had been disrupted. Poor alignment is, therefore, unlikely to be a primary feature of ICS, at least in the eight patients studied here, and may instead be caused by secondary effects such as viral infection, or even mechanical damage caused by coughing; both clinical symptoms of ICS (Afzelius,
ALIGNMENT
OF CILIA
IN IMMOTILE-CILIA
1979). Poor alignment of cilia has been correlated with respiratory tract infections (Holwell et al., 1980; Lungarella et al., 1983), and asthma (Laitinen et al., 1985). Furthermore, disorientated ciliary beat patterns have been described in the tracheobronchial tree of bronchitic rats (Iravani and Van As, 1972). We may conclude that alignment is likely to be worse among patients suffering from ICS because of their increased rate of infection, and that poor alignment in control 5 was a result of infection immediately before sampling. It might, therefore, be profitable to test alignment of cilia in ciliated cells from people with and without common infections. Some variation in ciliary alignment in different cells within the same epithelium might arise through sampling cells which are at different stages of their life-cycle. In nasal epithelia from embryonic mice, development of rootlets, basal feet and cilia precedes alignment (Frisch and Farbman, 1968), and in quail oviduct (Boisvieux-Ulrich et al., 1985) and rat lung (Sorokin, 1968) there is evidence that cilia are not aligned until they are mature and start beating. Sections through developing cells in normal epithelia might, therefore, show cilia that are poorly (randomly?) aligned. Furthermore, alignment might deteriorate with cell age, or at least deteriorate rapidly at cell death, thus producing a similar result, but in the absence of a method for measuring cell age this is difficult to establish. Afzelius (1981) suggested that basal bodies, unorientated when they migrate to the cell surface during development, are aligned by the act of beating, via the same hydromechanical forces that lead to metachronal coordination (see Machemer, 1975); if cilia are defective and unable to beat properly, as they often are in ICS, then alignment would not occur. This idea is difficult to test. In quail oviduct alignment is closely related, not only to the initiation of ciliary beating, but also to differentiation of the cortical
Fig. 3. Cross-sectioned Alignment was abnormally
cells from patient good. x22,5300.
527
SYNDROME
cytoskeleton, particularly of the microtubules and microfilaments associated with the basal feet (Boisvieux-Ulrich et al., 1985). If beating does help to align cilia then it may be a transient phenomenon with the basal bodies soon locked into place by the cortical cytoskeleton; it might even be the stimulus for cytoskeletal differentiation. Cilia on microsurgically reversed segments of rabbit oviduct maintain their original beat direction, against that of neighbouring cilia, for at least 13 months after the operation (Eddy et al., 1982), and cilia on fragments of cortex reversed through 180” in the protozoan Paramecium aurelia likewise maintain their original beat direction against that of neighbouring cilia, even through successive generations (Beisson and Sonneborn, 1965; Tamm et al., 1975). Assuming that the basal apparatus/cilium complex behaves as a unit, we may conclude that disruption of ciliary alignment involves general disruption of the cytoskeleton, which may affect alignment between neighbouring cells as well as between cilia within a cell. Many of the basal bodies in patient ‘A’ were arranged in rows, and alignment of the basal feet was particularly good, but the effects of this in terms of the function of the cilia is not clear. The arrangement, however, is unusual in human ciliated cells and we can assume that cytoskeletal elements normally not only help to anchor cilia with a suitable orientation, but also help to regulate their spatial distribution (see Reed et al., 1984). This process is not yet properly understood in any cell. Throughout this discussion we have assumed that the basal foot has a constant position relative to the ciliary axoneme, even in the patient group. This seems to be reasonable on the grounds that throughout the animal kingdom it has a predictable orientation relative to the ciliary effectivestroke, and that variation in basal foot alignment, as discussed above, is similar to that of
‘A’. The basal bodies
were arranged
in rows.
HOLLEY
the central microtubule pairs. The assumption could be tested by serial sectioning, particularly of cells in which alignment is very poor; until then, the possibility that the basal foot is mislocated on the basal body should not be ignored. Basal bodies sometimes support more than one basal foot (see Lungarella et al., 1985), thus illustrating the potential for a basal foot to be attached to a basal body at more than one site. In this report 1.1 and 2.6% of basal bodies, in controls and patients respectively, supported two&Sal feet. We may conclude that nasal cilia, in common with other mucus-propelling cilia, are not very accurately aligned. Alignment in patients suffering from ICS is rarely random, although generally less accurate than in controls, and it cannot reasonably be considered a primary feature of the disease. Poor align-
AND AFZELIlJS
ment is usually associated with a wide variety of other cellular and ciliary defects so it would not be easy to assess the effects of disorientation alone on mucus-propelling efficiency. There is little doubt, however, that poor alignment can only contribute to the problems of mucus clearance.
Acknowledgements
We thank those people who volunteered to undergo a nasal biopsy, Mrs Ulla Afzelius for patiently preparing sections, Drs R. W. Hiorns and Sophie Matthiesen for statistical advice, and Professor M. A. Sleigh for reading the manuscript. M.C.H. was supported by a Royal Society European Exchange Programme Fellowship.
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35