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Heterogeneity of ciliary morphology in the immotile-cilia syndrome in man
JOURNAL OF
ULTRASTRUCTURE
RESEARCH 73, 34--43 (1980)
Heterogeneity of Ciliary Morphology in the Immotile-Cilia Syndrome in Man E V E L I N E E . SCHNEEBERGER,* JOANNE MCCORMACK,* HENRY J. ISSENBERG,t SAMUEL R . SCHUSTER,~: AND PARK S. GERALD§
Departments of *Pathology, "~Cardiology, $Surgery and §Pediatrics, Children's Hospital Medical Center and Harvard Medical School, Boston, Massachusetts 02115 Received May 30, 1980 Detailed ultrastructural analysis of the respiratory cilia of three male patients with the immotilecilia syndrome is presented. They all had similar clinical manifestations, namely, situs inversus totalis, repeated respiratory infections, and sinusitis. The respiratory cilia, however, of these patients showed a variety of structural abnormalities some of which are described for the first time. In the cilia of the first patient both the dynein arms were lacking, and, almost 40% of the cilia had one to nine supernumerary microtubules peripheral to the nine outer doublets. The cilia of the second patient showed an absence of inner dynein arms, unusually prominent outer dynein arms, a spoke defect in 46% of cilia examined, and no morphologically detectable nexin links. The cilia of the last patient lacked both dynein arms but, in contrast to the first two patients, showed virtually no microtubular abnormalities. These patients demonstrate that a variety of structural abnormalities in respiratory cilia can result in virtually identical clinical manifestations.
(Sturgess et al., 1979a), absence of the central pair of microtubules with transposition of the number one outer microtubule doublet to the center (Sturgess et al., 1979b, 1980), absence of either outer or inner dynein arms, or both, and absence of the central sheath (Afzelius and Eliasson, 1979). In the course of an ultrastructural study of nasal cilia from patients with cardiac malrotation (Gerald et al., 1978), we examined the cilia of three patients who had the clinical features of the immotile-cilia syndrome. While one of them exhibited the absence of both dynein arms, the remaining two exhibited unusual ultrastructural features which have so far not been described in the immotile-cilia syndrome. The observations made in these three patients are the basis for the present study.
In 1933 Kartagener documented the association of bronchiectasis and sinusitis with situs inversus (Kartagener, 1933). New insight into the pathogenesis of this disorder was provided by the demonstration that the sperm of infertile men with Kartagener's syndrome were immotile and lacked the dynein arms normally attached to the outer microtubular doublets (Afzelius et al., 1975, Pedersen and Rebbe, 1975). Respiratory tract cilia from such patients were shown to have a similar dynein arm defect and to exhibit r e duc e d motility (Afzelius, 1976; Pedersen and Mygind, 1976). Since not all patients with immotile cilia have situs inversus, it has been suggested that the clinical consequences of this ciliary defect be reclassified as "the iramotile-cilia s y n d r o m e " (Eliasson et al., 1977), and to include those with the Kartagener's triad as a subgroup. Recent morphological studies have shown that the immotile-cilia syndrome may be a consequence of a variety of other axonemal, ultrastructural defects (Afzelius and Eliasson, 1979; Sturgess et al., 1979a,b, 1980). These include ciliary spoke defects
MATERIALS AND METHODS Nasal brush biopsies were obtained from three patients (JC, GB, and GJ) with the clinical features of the immotile-cilia syndrome, as well as from both parents of GB. A nasal brush biopsy from a normal 6-year-old child was used as a control. As indicated in Table I the patients ranged in age from 4 to 34 years and all three of them were males. They all had situs 34
0022-5320/80/100034-10502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
IMMOTILE-CILIA SYNDROME
35
TABLE I AGE, CLINICAL MANIFESTATION, AND CILIARY MORPHOLOGY OF THREE MALE PATIENTS WITH IMMOT1LECILIA SYNDROME
Abnormalities in microtubular organization (%)a PaAge tient ( y e a r s ) JC
4
GB
GJ
Clinical abnormalities
No. of cilia examined
Dyneinarms
SIT, recurrent respiratory infections, sinusitis, otitis media, pectus excavatum
241
Partially or completely absent
34
SIT, recurrent respiratory infections, sinusitis, otitis media, oligospermia and immotile sperm
605
Prominent outer and absent inner arms
16
SIT, recurrent respiratory infections, sinusitis
197
Absent
9+ 2 + xb
9+(0 or 4) + yr.
38.0
5.0
10
( 9 - n) + 2 + z'~ 9 + 2E'~ 2.0
30.0
16.0
1.0
Note. SIT: situs inversus totalis. In none of the 154 cilia examined from a normal 6-year-old girl were any of the above microtubule abnormalities observed. The first figure indicates the number of outer doublets, the second figure the number of central microtubules, and the third figure the number of supernumerary microtubules peripheral or interior to the outer ring of doublets. ~' Cilia with supernumerary peripheral microtubules, x = 1-9. c Cilia with either an absence or duplication of the central microtubular pair. y = 0-2 supernumerary single central microtubules. ,/Cilia with nine or fewer (n = 0-2) outer doublets, and central displacement Of (z = 1-2) outer doublets. The notation 2E indicates the eccentric placement of the two central microtubules.
inversus totalis (SIT), recurrent respiratory infections, and sinusitis, and two of them (GB and JC) had recurrent otitis media. JC also had pectus excavatum. The family history was negative in both JC and GJ, but GB had a sibling who died at 9 months of age of congenital heart disease. No further anatomical information is available on the kind of cardiac malformation that was present. Semen obtained from the oldest male (GB) as well as from his father was examined for sperm motility, and a portion was processed for electron microscopy. Nasopharyngeal cilia were obtained from these patients under local anesthesia by means of nasal brush biopsy procedure. The mucosal specimen was immersed for 2 hr in formaldehyde-glutaraldehyde fixative (Karnovsky, 1965), diluted 1:2 with 0.1 M cacodylate buffer, pH 7.3, at 4°C, and washed overnight in 0.15 M cacodylate buffer, pH 7.3. Small fragments of tissue were then removed from the bristles of the brush, postfixed in 2% aqueous osmium tetroxide, stained with uranyl acetate, dehydrated in graded alcohol, and embedded in Epon. Thin sections, stained
with lead citrate, were examined in a Philips 300 electron microscope. RESULTS
Technical Considerations The
nasopharyngeal
clinically valuable
brush
tissue
biopsy
sampling
is a tech-
n i q u e i n t h a t it c a n b e d o n e o n a n o u t p a t i e n t basis, even on small children. This enables the physician to detect ciliary abnormalities v e r y e a r l y in life, a n d to t a k e m e a s u r e s to ameliorate the pulmonary the immotile-cilia
complications of
syndrome.
In over
25
biopsies examined by the authors, only one f a i l e d t o y i e l d a n y cilia. I n a s s e s s i n g o v e r a l l ciliary o r i e n t a t i o n in n a s a l b r u s h b i o p s y m a t e r i a l , h o w e v e r , s o m e c a u t i o n is n e c e s s a r y , s i n c e a n a r t i f a c t u a l c h a n g e in orien-
36
SCHNEEBERGER ET AL.
Fir. 1. Nasal brush biopsy from a normal individual showing five similarly oriented cilia as judged by the alignment Of the two central microtubules (CM). The nine outer doublets are composed of A and B microtubules. The outer (OD) and inner (ID) dynein arms are attached to the A tubules. The central sheath (S), spokes (Sp), and nexin links (N) are indicated, x 142 500.
r a t i o n m a y o c c u r as the cilia b e c o m e enm e s h e d in the bristles of the b r u s h . T h i s c a n b e s t be c i r c u m v e n t e d b y e i t h e r (a) ass e s s i n g the o r i e n t a t i o n o f the two c e n t r a l m i c r o t u b u l e s i n n e i g h b o r i n g cilia i n sect i o n s c u t close to the cell b o d y (Fig. 1) or (b) a s s e s s i n g the o r i e n t a t i o n of the l a t e r a l s p u r s a t t a c h e d to the b a s a l b o d i e s (Fig. 2). T h e f o l l o w i n g t h r e e p a t i e n t s all h a d the clinical f e a t u r e s of the i m m o t i l e - c i l i a synd r o m e , h o w e v e r , t h e i r cilia d e m o n s t r a t e d a variety of structural abnormalities, some
of w h i c h h a v e h i t h e r t o n o t b e e n d e s c r i b e d in r e s p i r a t o r y cilia.
Absence of Inner and Outer Dynein Arms and Presence of Supernumerary Outer Microtubules I n the b r u s h b i o p s y of the y o u n g e s t pat i e n t JC ( T a b l e I), ciliated cells w e r e easily identified, a l t h o u g h , t h o s e p r e s e n t h a d dec r e a s e d n u m b e r s of cilia. A total of 241 cilia w e r e e x a m i n e d . I n all of t h e m t h e r e was a c o m p l e t e or partial a b s e n c e of b o t h i n n e r
FIG. 2. Sections of ciliated cells cut at the level of the basal bodies. (a) Cell from a normal individual. (b) Cell from patient JC whose cilia lack dynein arms and have many supernumerary microtubules. (c) Cell from patient GB the cilia of whom lack inner dynein arms and nexin links and have a partial spoke defect. (d) Cell from patient GJ whose cilia lack inner and outer dynein arms. Note that the spurs (arrows) attached to the basal bodies of the normal control (a) are all oriented in the same direction. By contrast, the spurs on those from the three patients (b-d) show a random orientation (arrows). x 40 000.
IMMOTILE-CILIA SYNDROME
37
38
S C H N E E B E R G E R ET AL.
FIG. 3. (a) Cross section of cilia from patient JC. In this field five of seven cilia have three to seven supernumerary outer single microtubules (small lines). (b) Longitudinal section of a cilium from patient JC showing one of the supernumerary outer microtubules (arrow). (c,d) High magnification of cilia from patient JC. Dynein arms are absent in cilia regardless of whether they have (c) or lack (d) supernumerary microtubules. Nexin links (N) are present. (a), x 122 000; (b), x 100 000; (c) and (d), x 252 000.
IMMOTILE-CILIA SYNDROME
39
P
b
{
c
Fie. 4. Cilia from patient GB. All cilia observed have prominent outer (OD) but no inner dynein arms. No nexin links were seen in any of the cilia examined. (a) One of the cilia in which the two central microtubules (CM) are eccentrically placed and an outer microtubular doublet (AB) is displaced centrally. The spokes (Sp) appear disorganized. (b) One of the cilia in which the central microtubules (CM) are eccentrically placed and the spokes (Sp) are disorganized. However, the nine outer doublets form the usual outer ring. (c) One of the cilia from patient GB which, except for prominent outer (OD) and absent inner dynein arms, and no discernible nexin links, has normally placed microtubules and evenly spaced spokes (Sp). (d) One of the rare cilia observed in patient GB in which the two central microtubules (CM) are duplicated and a normal number of outer doublets are present. (a)-(c), x 195 000; (d), × 160 000.
a n d o u t e r d y n e i n a r m s , a l t h o u g h , the n e x i n links, spokes, spoke heads, and central sheaths were clearly discernible (Figs. 3c,d). M a n y o f t h e cilia w e r e r a n d o m l y o r i e n t e d as j u d g e d b y the a l i g n m e n t of the two c e n t r a l m i c r o t u b u l e s , as well as b y the
r a n d o m o r i e n t a t i o n o f the s p u r s a t t a c h e d to the b a s a l b o d i e s of the cilia (Fig. 2a). T h e m o s t r e m a r k a b l e f e a t u r e i n this b i o p s y , h o w e v e r , was the p r e s e n c e , in 38% of the cilia e x a m i n e d , of o n e to n i n e s u p e r n u m e r ary single m i c r o t u b u l e s a r r a n g e d p e r i p h e r a l
40
SCHNEEBERGER ET AL.
to the nine outer doublets (Figs. 3a,b). In an additional 5% of the cilia there was either an absence of the two central microtubules or one to two supernumerary single microtubules were present internal to the nine outer doublets. Finally, in 2% of the cilia fewer than nine outer doublets were present.
Absence of Inner Dynein Arm, Absence of Nexin Links, and Partial Spoke Defect T w o nasal b r u s h b i o p s i e s , s p a c e d 3 months apart, were examined from patient GB. A total of 605 cilia were examined, and in both samples similar structural abnormalities were observed. In all cilia there was a lack of the inner dynein arms, with the outer ones being unusually prominent, and no discernible nexin links (Figs. 4a-d). In 44% of the cilia the nine outer doublets were concentrically arranged around the two central microtubules and spokes connecting the central sheath with the outer doublets were clearly seen. In another 30% of the cilia the spokes appeared disorganized, the two central microtubules were eccentrically placed, and one of the outer doublets was displaced centrally (Fig. 4a). In still another 16% of the cilia the spokes appeared similarly disorganized, the central microtubules were in an eccentric position with respect to the nine outer doublets, but none of the latter were displaced centrally (Fig. 4b). These abnormalities in microtubular arrangement were more apparent in the distal portions of the cilia than near their bases. Lateral views of the cilia clearly showed a similar abnormal placement of these microtubular elements. In 10% of cilia other abnormalities of microtubular arrangements were observed. These included either a duplication of the central microtubular pair (Fig. 4d) or their complete absence. In some cilia there was the presence of one or two microtubular doublets within the confines of the normal 9 + 2 microtubular configuration. Ejaculates from patient GB and his father were examined for sperm number and mo-
tility, and a sample from both individuals was examined by electron microscopy by Dr. Don Fawcett. The sperm of GB were greatly reduced in number and all were immotile. The ejaculate examined by electron microscopy yielded only cell debris and epithelial cells and no spermatozoa were identified. The sperm count of the patient's father was within normal limits and motility was approximately 50% of normal. By electron microscopy there were increased numbers of sperm with axonemal abnormalities which included chiefly a reduction in the number of microtubules, but all sperm had morphologically recognizable dynein arms. Nasal brush biopsies from both parents of GB were also examined. Of the 104 cilia examined from the mother, only 3% were abnormal, and had an 8 + 2 microtubular configuration. In the sample obtained from the father 223 cilia were examined. Of these 9% were abnormal, and showed an 8 + 2, 7 + 2, or 6 + 2 microtubular configuration. The cilia from both parents were otherwise morphologically normal.
Absence of Inner and Outer Dynein Arms In the nasal brush biopsy of patient GJ with classical K a r t a g e n e r ' s s y n d r o m e , there was a reduction in the number of cilia. These were randomly oriented as assessed by the orientation of the spurs attached to the basal bodies, as well as the two central microtubules (Figs. 2b, 5). A total of 197 cilia w e r e e x a m i n e d , and all o f t h e m showed a complete absence of both dynein arms (Fig. 5). Nexin links, spokes, spoke heads, and central sheaths were clearly discernible (Fig. 5). In contrast to the first two cases, virtually no abnormalities in microtubular configuration were seen. In fact only two cilia showed any microtubular abnormality, and this was in the form of either a 7 + 2 configuration or a 9 + 2 + 1 configuration, in which a single extra microtubule was present within the confines of the outer ring of nine doublets. This frequency of tubular abnormalities was similar to that observed in the normal control patient in
41
IMMOTILE-CILIA SYNDROME
4
N '
s
FIG. 5. Cross section of five cilia from patient GJ. Note that the central microtubules are randomly oriented. I n n e r and outer dynein arms are absent, but nexin links (N), as well as other ciliary structures (spokes, central sheath), are clearly visible, x 151 000.
which 1.5%, of 154 cilia examined, were abnormal. The abnormal cilia were of the compound variety, i.e., two sets of 9 + 2 axonemal structures were present within the same cell membrane. DISCUSSION
The early studies of Gibbons (1965) and Satir (1965) have clearly shown that the inner and outer dynein arms correspond to an ATPase which is essential in generating an active sliding motion of one neighboring microtubular doublet relative to its neighbor. That the absence of one or both of the dynein arms results in partial or complete axonemal paralysis has been amply dem-
onstrated in humans (Afzelius et al., 1975; Afzelius, 1976; Afzelius and Eliasson, 1979), mice (Bryan, 1977; Bryan et al., 1977; Bryan and Chandler, 1978), and a paralyzed flagellar (pf) mutant of a Chlamydom o n a s strain (Huang et al., 1978). More recently, it has become apparent from experiments using a variety of pf mutants of C h l a m y d o m o n a s (Randall and Starling, 1972; Piperno et al., 1977; Adams et al., 1978; Witman et al., 1978), as well as from the examination of human material (Sturgess et al., 1979b, 1980; Afzelius and Eliasson, 1979), that the absence of other axonemal structures such as spoke heads and/ or spokes, one or both central microtubules, central microtubules, and central
42
S C H N E E B E R G E R ET AL.
sheath complex also results in flagellar paralysis. These observations support the studies of Warner and Satir (1974), who showed that components of the central sheath and/or spokes are essential for the conversion of interdoublet sliding into axonemal bending. The present study reports on several structural abnormalities in cilia from patients with the immotile-cilia syndrome which have thus far not been described, and demonstrates further that a large variety of structural abnormalities of respiratory cilia are associated with virtually identical clinical manifestations. The respiratory cilia of the first patient, JC, while lacking both dynein arms, were further abnormal in that 38% of the cilia contained one to nine supernumerary microtubules arranged external to the nine outer doublets. The effect that supernumerary microtubules might have on ciliary function is not known. It is conceivable that the presence of such extra microtubules, which are not connected to axonemal structures that are known to participate in the coordinated movement of cilia (Gibbons, 1976), might further impair ciliary function. Abnormalities in microtubular number and arrangement have been observed in patients with Kartagener's syndrome (Katz et al., 1977; Afzelius, 1977), chronic bronchitis (Ailsby and Ghadially, 1973; Konradova et al., 1975; McDowell et al., 1976), and lung carcinoma (McDowell et al., 1976). However, in none of these patients did the cilia with abnormal microtubular formations constitute more than 5% of the cilia examined. Of interest is a recent report in which as many as 24% of respiratory cilia of patients with retinitis pigmentosa were observed to have a variety of microtubular malformations (Arden and Fox, 1979). While the underlying mechanism responsible for the presence of supernumerary microtubules in such a large number of cilia in patient JC is unclear, it is probably not due to the chronic infections which are a prominent feature of the immotile-cilia syndrome. Being the young-
est patient, in the present series, JC had not experienced as many respiratory infections as the remaining two patients (GB and GJ) whose cilia did not have supernumerary microtubules. Furthermore, the cilia of GJ, which have the morphological characteristics of classical Kartagener's syndrome, were remarkably free of any microtubular abnormalities. The cilia of patient GB similarly demonstrated a number of unusual structural abnormalities. Forty-six percent of his respiratory cilia had the morphological features associated with a spoke defect; that is, eccentric central microtubules, and the displacement of one of the outer doublets toward the center. This finding differs from that originally described by Sturgess et al. (1979a) in which 100% of the cilia showed this shift in microtubular placement. Furthermore, in the three patients with the spoke defect which were examined by Sturgess and her colleagues, both dynein arms as well as nexin links were said to be present. In GB the inner dynein arms were absent and the outer ones were unusually prominent. Nexin links were not seen in any of the cilia from patient GB. Although nexin links may be difficult to resolve, they were readily seen in both of the other two patients (JC, G J) as well as in the normal control. Thus far no pf mutant of C h l a m y d o m o nas has been described in which there is an absence of nexin links. Indirect evidence gained from pf mutants which lack radial spokes, but in which nexin links are present (Witman et al., 1978), suggests that nexin finks form permanent bridges between adjacent outer doublets, and may, therefore, serve to maintain the structural integrity of the axoneme against dynein-generated shear forces. The present observations in patient GB are somewhat surprising in this regard, since more complete outer doublet disarray might be expected in cilia which lack nexin links and also have a spoke defect. It is conceivable that the outer dynein arms act, at least in part, to keep the outer
IMMOTILE-CILIA SYNDROME
doublets in place and are able to do so even in the absence of nexin links. While the purpose of the present study was to examine in detail the different structural abnormalities in three patients with the immotile-cilia syndrome, it is of interest to note that the father of GB had a moderate number (9% of cilia examined) of abnormal respiratory cilia, as well as some abnormal sperm. Furthermore, GB had a sibling who died at 9 months of congenital heart disease. Although Kartagener's syndrome, as defined by the clinical triad of situs inversus totalis, bronchiectasis, and sinusitis, is thought to be inherited as an autosomal recessive (Rott, 1979), the mode of inheritance of the various structural abnormalities of cilia which give rise to the immotile-cilia syndrome remains to be established. This work was supported by USPHS Grants HL25822, HL-07193, and HD-04807 and by Grant RR 128 from the General Clinical Research Centers Program of the Division of Research Resources of the National Institutes of Health. The examination of the sperm samples by Dr. Don Fawcett and the clinical coordination by Mrs. Pamela Hawley are gratefully acknowl-
edged. REFERENCES ADAMS, G. M. W., HUANG, B., PIPERNO, G., AND LUCK, D. J. L. (1978) J. Cell Biol. 79, 280a. AFZELIUS, B. A. (1976) Science 193, 317-319. AFZELIUS, B. A. (1977) N. Engl. J. Med. 297, 1012. AFZELIUS, B. A., AND ELIASSON,R. (1979) J. Ultrastruct. Res. 69, 43-52. AFZEEIUS, B. A., ELIASSON, R., JOHNSEN, O., AND LINDHOEMER, C. (1975) J. Cell Biol. 66, 224-232. AILSBY, R. L., AND GHADIALLY,F. N. (1973) J. Pathol. 109, 75-78. ARDEN, G. B., AND FOX, B. (1979) Nature (London) 279, 534-536. BRYAN, J. H. D. (1977) Cell Tissue Res. 180, 187201.
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BRYAN, J. H. D., BATES, T. J., AND CHANDLER, D. B. (1977) Excerpta Med. Found. Int. Congr. Set. 426, 52. BRYAN, J. H. D., AND CHANDLER, O. B. (1978) J. Cell Biol. 79, 281a. ELIASSON, R., MOSSBERG, B., CAMNER, P., AND AFZELIUS, B. A. (1977) N. Engl. J. Med. 297, 1-6. GERALD, P. S., SCHUSTER, S. R., RUCKMAN, R. N., AND SCHNEEBERGER,E. E. (1978) Pediat. Res. 12, 515. GIBBONS, I. R. (1965) Arch. Biol. 76, 317-352. GIBBONS, I. R. (1976) in BRINKLEY, B. R., AND PORTER, K. R. (Eds.), International Cell Biology, pp. 348-357, Rockefeller Press, New York. HUANG, B., PrPERNO, G., AND LUCK, D. J. L. (1978) J. Cell Biol. 79, 286a. KARNOVSKV, M. J. (1965) J. Cell Biol. 27, 137. KARTAGENER,M. (1933) Beitr. Klin. Tuberk. 83, 489501. KATZ, S. M., DAMJANOV, I., CARVER, J., SPITZER, S., MASON, D., KAUFFMAN, L. A., AND SOLNICK, P. (1977) N. Engl. J. Med. 297, 1011-1012. KONRADOVA, V., HLOUSKOVA, Z., AND TOMANEK, A. (1975) Folia Morphol. 23, 293-295. McDowELL, E. M., BARRETT, L. A., HARRIS,C. C., AND TRUMP, B. F. (1976) Arch. Pathol. Lab. Med. 100, 429-436. PEDERSEN, H., AND MYGIND, N. (1976) Nature (London) 262, 494495. PEDERSEN, H., AND REBBE, H. (1975) Biol. Reprod. 12, 541-544. PIPERNO, G., HUANG, G., AND LUCK, D. J. L. (1977) Proc. Nat. Acad. Sci. USA 74, 1600-1604. RANDALL, J., AND STARLING, D. (1972) in BEATTY, R. A., AND GLUECKSOHN-WAELSCH,S. (Eds.), The Genetics of the Spermatozoon, pp. 13-36, University of Edinburgh, Edinburgh. ROTT, H. D. (1979) Hum. Genet. 46, 249-261. SATIR, P. (1965) J. Cell Biol. 26, 805-834. STURGESS, J. M., CHAO, J., AND TURNER, J. A. P, (1979b) J. Cell Biol. 83, 179a. STURGESS, J. M., CHAO, J., AND TURNER, J. A. P. (1980) N. Engl. J. Med. 303, 318. STURGESS, J. M., CHAO, J., WONG, J., ASPIN, N., AND TURNER, J. A. P. (1979a) N. Engl. J. Med. 300, 53-56. WARNER, F. D., AND SATIR, P. (1974) J. Cell Biol. 63, 35-63. WITMAN, G. B., PEUMMER, J., AND SANDER, G. (1978) J. Cell Biol. 76, 729-747.
ULTRASTRUCTURE
RESEARCH 73, 34--43 (1980)
Heterogeneity of Ciliary Morphology in the Immotile-Cilia Syndrome in Man E V E L I N E E . SCHNEEBERGER,* JOANNE MCCORMACK,* HENRY J. ISSENBERG,t SAMUEL R . SCHUSTER,~: AND PARK S. GERALD§
Departments of *Pathology, "~Cardiology, $Surgery and §Pediatrics, Children's Hospital Medical Center and Harvard Medical School, Boston, Massachusetts 02115 Received May 30, 1980 Detailed ultrastructural analysis of the respiratory cilia of three male patients with the immotilecilia syndrome is presented. They all had similar clinical manifestations, namely, situs inversus totalis, repeated respiratory infections, and sinusitis. The respiratory cilia, however, of these patients showed a variety of structural abnormalities some of which are described for the first time. In the cilia of the first patient both the dynein arms were lacking, and, almost 40% of the cilia had one to nine supernumerary microtubules peripheral to the nine outer doublets. The cilia of the second patient showed an absence of inner dynein arms, unusually prominent outer dynein arms, a spoke defect in 46% of cilia examined, and no morphologically detectable nexin links. The cilia of the last patient lacked both dynein arms but, in contrast to the first two patients, showed virtually no microtubular abnormalities. These patients demonstrate that a variety of structural abnormalities in respiratory cilia can result in virtually identical clinical manifestations.
(Sturgess et al., 1979a), absence of the central pair of microtubules with transposition of the number one outer microtubule doublet to the center (Sturgess et al., 1979b, 1980), absence of either outer or inner dynein arms, or both, and absence of the central sheath (Afzelius and Eliasson, 1979). In the course of an ultrastructural study of nasal cilia from patients with cardiac malrotation (Gerald et al., 1978), we examined the cilia of three patients who had the clinical features of the immotile-cilia syndrome. While one of them exhibited the absence of both dynein arms, the remaining two exhibited unusual ultrastructural features which have so far not been described in the immotile-cilia syndrome. The observations made in these three patients are the basis for the present study.
In 1933 Kartagener documented the association of bronchiectasis and sinusitis with situs inversus (Kartagener, 1933). New insight into the pathogenesis of this disorder was provided by the demonstration that the sperm of infertile men with Kartagener's syndrome were immotile and lacked the dynein arms normally attached to the outer microtubular doublets (Afzelius et al., 1975, Pedersen and Rebbe, 1975). Respiratory tract cilia from such patients were shown to have a similar dynein arm defect and to exhibit r e duc e d motility (Afzelius, 1976; Pedersen and Mygind, 1976). Since not all patients with immotile cilia have situs inversus, it has been suggested that the clinical consequences of this ciliary defect be reclassified as "the iramotile-cilia s y n d r o m e " (Eliasson et al., 1977), and to include those with the Kartagener's triad as a subgroup. Recent morphological studies have shown that the immotile-cilia syndrome may be a consequence of a variety of other axonemal, ultrastructural defects (Afzelius and Eliasson, 1979; Sturgess et al., 1979a,b, 1980). These include ciliary spoke defects
MATERIALS AND METHODS Nasal brush biopsies were obtained from three patients (JC, GB, and GJ) with the clinical features of the immotile-cilia syndrome, as well as from both parents of GB. A nasal brush biopsy from a normal 6-year-old child was used as a control. As indicated in Table I the patients ranged in age from 4 to 34 years and all three of them were males. They all had situs 34
0022-5320/80/100034-10502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
IMMOTILE-CILIA SYNDROME
35
TABLE I AGE, CLINICAL MANIFESTATION, AND CILIARY MORPHOLOGY OF THREE MALE PATIENTS WITH IMMOT1LECILIA SYNDROME
Abnormalities in microtubular organization (%)a PaAge tient ( y e a r s ) JC
4
GB
GJ
Clinical abnormalities
No. of cilia examined
Dyneinarms
SIT, recurrent respiratory infections, sinusitis, otitis media, pectus excavatum
241
Partially or completely absent
34
SIT, recurrent respiratory infections, sinusitis, otitis media, oligospermia and immotile sperm
605
Prominent outer and absent inner arms
16
SIT, recurrent respiratory infections, sinusitis
197
Absent
9+ 2 + xb
9+(0 or 4) + yr.
38.0
5.0
10
( 9 - n) + 2 + z'~ 9 + 2E'~ 2.0
30.0
16.0
1.0
Note. SIT: situs inversus totalis. In none of the 154 cilia examined from a normal 6-year-old girl were any of the above microtubule abnormalities observed. The first figure indicates the number of outer doublets, the second figure the number of central microtubules, and the third figure the number of supernumerary microtubules peripheral or interior to the outer ring of doublets. ~' Cilia with supernumerary peripheral microtubules, x = 1-9. c Cilia with either an absence or duplication of the central microtubular pair. y = 0-2 supernumerary single central microtubules. ,/Cilia with nine or fewer (n = 0-2) outer doublets, and central displacement Of (z = 1-2) outer doublets. The notation 2E indicates the eccentric placement of the two central microtubules.
inversus totalis (SIT), recurrent respiratory infections, and sinusitis, and two of them (GB and JC) had recurrent otitis media. JC also had pectus excavatum. The family history was negative in both JC and GJ, but GB had a sibling who died at 9 months of age of congenital heart disease. No further anatomical information is available on the kind of cardiac malformation that was present. Semen obtained from the oldest male (GB) as well as from his father was examined for sperm motility, and a portion was processed for electron microscopy. Nasopharyngeal cilia were obtained from these patients under local anesthesia by means of nasal brush biopsy procedure. The mucosal specimen was immersed for 2 hr in formaldehyde-glutaraldehyde fixative (Karnovsky, 1965), diluted 1:2 with 0.1 M cacodylate buffer, pH 7.3, at 4°C, and washed overnight in 0.15 M cacodylate buffer, pH 7.3. Small fragments of tissue were then removed from the bristles of the brush, postfixed in 2% aqueous osmium tetroxide, stained with uranyl acetate, dehydrated in graded alcohol, and embedded in Epon. Thin sections, stained
with lead citrate, were examined in a Philips 300 electron microscope. RESULTS
Technical Considerations The
nasopharyngeal
clinically valuable
brush
tissue
biopsy
sampling
is a tech-
n i q u e i n t h a t it c a n b e d o n e o n a n o u t p a t i e n t basis, even on small children. This enables the physician to detect ciliary abnormalities v e r y e a r l y in life, a n d to t a k e m e a s u r e s to ameliorate the pulmonary the immotile-cilia
complications of
syndrome.
In over
25
biopsies examined by the authors, only one f a i l e d t o y i e l d a n y cilia. I n a s s e s s i n g o v e r a l l ciliary o r i e n t a t i o n in n a s a l b r u s h b i o p s y m a t e r i a l , h o w e v e r , s o m e c a u t i o n is n e c e s s a r y , s i n c e a n a r t i f a c t u a l c h a n g e in orien-
36
SCHNEEBERGER ET AL.
Fir. 1. Nasal brush biopsy from a normal individual showing five similarly oriented cilia as judged by the alignment Of the two central microtubules (CM). The nine outer doublets are composed of A and B microtubules. The outer (OD) and inner (ID) dynein arms are attached to the A tubules. The central sheath (S), spokes (Sp), and nexin links (N) are indicated, x 142 500.
r a t i o n m a y o c c u r as the cilia b e c o m e enm e s h e d in the bristles of the b r u s h . T h i s c a n b e s t be c i r c u m v e n t e d b y e i t h e r (a) ass e s s i n g the o r i e n t a t i o n o f the two c e n t r a l m i c r o t u b u l e s i n n e i g h b o r i n g cilia i n sect i o n s c u t close to the cell b o d y (Fig. 1) or (b) a s s e s s i n g the o r i e n t a t i o n of the l a t e r a l s p u r s a t t a c h e d to the b a s a l b o d i e s (Fig. 2). T h e f o l l o w i n g t h r e e p a t i e n t s all h a d the clinical f e a t u r e s of the i m m o t i l e - c i l i a synd r o m e , h o w e v e r , t h e i r cilia d e m o n s t r a t e d a variety of structural abnormalities, some
of w h i c h h a v e h i t h e r t o n o t b e e n d e s c r i b e d in r e s p i r a t o r y cilia.
Absence of Inner and Outer Dynein Arms and Presence of Supernumerary Outer Microtubules I n the b r u s h b i o p s y of the y o u n g e s t pat i e n t JC ( T a b l e I), ciliated cells w e r e easily identified, a l t h o u g h , t h o s e p r e s e n t h a d dec r e a s e d n u m b e r s of cilia. A total of 241 cilia w e r e e x a m i n e d . I n all of t h e m t h e r e was a c o m p l e t e or partial a b s e n c e of b o t h i n n e r
FIG. 2. Sections of ciliated cells cut at the level of the basal bodies. (a) Cell from a normal individual. (b) Cell from patient JC whose cilia lack dynein arms and have many supernumerary microtubules. (c) Cell from patient GB the cilia of whom lack inner dynein arms and nexin links and have a partial spoke defect. (d) Cell from patient GJ whose cilia lack inner and outer dynein arms. Note that the spurs (arrows) attached to the basal bodies of the normal control (a) are all oriented in the same direction. By contrast, the spurs on those from the three patients (b-d) show a random orientation (arrows). x 40 000.
IMMOTILE-CILIA SYNDROME
37
38
S C H N E E B E R G E R ET AL.
FIG. 3. (a) Cross section of cilia from patient JC. In this field five of seven cilia have three to seven supernumerary outer single microtubules (small lines). (b) Longitudinal section of a cilium from patient JC showing one of the supernumerary outer microtubules (arrow). (c,d) High magnification of cilia from patient JC. Dynein arms are absent in cilia regardless of whether they have (c) or lack (d) supernumerary microtubules. Nexin links (N) are present. (a), x 122 000; (b), x 100 000; (c) and (d), x 252 000.
IMMOTILE-CILIA SYNDROME
39
P
b
{
c
Fie. 4. Cilia from patient GB. All cilia observed have prominent outer (OD) but no inner dynein arms. No nexin links were seen in any of the cilia examined. (a) One of the cilia in which the two central microtubules (CM) are eccentrically placed and an outer microtubular doublet (AB) is displaced centrally. The spokes (Sp) appear disorganized. (b) One of the cilia in which the central microtubules (CM) are eccentrically placed and the spokes (Sp) are disorganized. However, the nine outer doublets form the usual outer ring. (c) One of the cilia from patient GB which, except for prominent outer (OD) and absent inner dynein arms, and no discernible nexin links, has normally placed microtubules and evenly spaced spokes (Sp). (d) One of the rare cilia observed in patient GB in which the two central microtubules (CM) are duplicated and a normal number of outer doublets are present. (a)-(c), x 195 000; (d), × 160 000.
a n d o u t e r d y n e i n a r m s , a l t h o u g h , the n e x i n links, spokes, spoke heads, and central sheaths were clearly discernible (Figs. 3c,d). M a n y o f t h e cilia w e r e r a n d o m l y o r i e n t e d as j u d g e d b y the a l i g n m e n t of the two c e n t r a l m i c r o t u b u l e s , as well as b y the
r a n d o m o r i e n t a t i o n o f the s p u r s a t t a c h e d to the b a s a l b o d i e s of the cilia (Fig. 2a). T h e m o s t r e m a r k a b l e f e a t u r e i n this b i o p s y , h o w e v e r , was the p r e s e n c e , in 38% of the cilia e x a m i n e d , of o n e to n i n e s u p e r n u m e r ary single m i c r o t u b u l e s a r r a n g e d p e r i p h e r a l
40
SCHNEEBERGER ET AL.
to the nine outer doublets (Figs. 3a,b). In an additional 5% of the cilia there was either an absence of the two central microtubules or one to two supernumerary single microtubules were present internal to the nine outer doublets. Finally, in 2% of the cilia fewer than nine outer doublets were present.
Absence of Inner Dynein Arm, Absence of Nexin Links, and Partial Spoke Defect T w o nasal b r u s h b i o p s i e s , s p a c e d 3 months apart, were examined from patient GB. A total of 605 cilia were examined, and in both samples similar structural abnormalities were observed. In all cilia there was a lack of the inner dynein arms, with the outer ones being unusually prominent, and no discernible nexin links (Figs. 4a-d). In 44% of the cilia the nine outer doublets were concentrically arranged around the two central microtubules and spokes connecting the central sheath with the outer doublets were clearly seen. In another 30% of the cilia the spokes appeared disorganized, the two central microtubules were eccentrically placed, and one of the outer doublets was displaced centrally (Fig. 4a). In still another 16% of the cilia the spokes appeared similarly disorganized, the central microtubules were in an eccentric position with respect to the nine outer doublets, but none of the latter were displaced centrally (Fig. 4b). These abnormalities in microtubular arrangement were more apparent in the distal portions of the cilia than near their bases. Lateral views of the cilia clearly showed a similar abnormal placement of these microtubular elements. In 10% of cilia other abnormalities of microtubular arrangements were observed. These included either a duplication of the central microtubular pair (Fig. 4d) or their complete absence. In some cilia there was the presence of one or two microtubular doublets within the confines of the normal 9 + 2 microtubular configuration. Ejaculates from patient GB and his father were examined for sperm number and mo-
tility, and a sample from both individuals was examined by electron microscopy by Dr. Don Fawcett. The sperm of GB were greatly reduced in number and all were immotile. The ejaculate examined by electron microscopy yielded only cell debris and epithelial cells and no spermatozoa were identified. The sperm count of the patient's father was within normal limits and motility was approximately 50% of normal. By electron microscopy there were increased numbers of sperm with axonemal abnormalities which included chiefly a reduction in the number of microtubules, but all sperm had morphologically recognizable dynein arms. Nasal brush biopsies from both parents of GB were also examined. Of the 104 cilia examined from the mother, only 3% were abnormal, and had an 8 + 2 microtubular configuration. In the sample obtained from the father 223 cilia were examined. Of these 9% were abnormal, and showed an 8 + 2, 7 + 2, or 6 + 2 microtubular configuration. The cilia from both parents were otherwise morphologically normal.
Absence of Inner and Outer Dynein Arms In the nasal brush biopsy of patient GJ with classical K a r t a g e n e r ' s s y n d r o m e , there was a reduction in the number of cilia. These were randomly oriented as assessed by the orientation of the spurs attached to the basal bodies, as well as the two central microtubules (Figs. 2b, 5). A total of 197 cilia w e r e e x a m i n e d , and all o f t h e m showed a complete absence of both dynein arms (Fig. 5). Nexin links, spokes, spoke heads, and central sheaths were clearly discernible (Fig. 5). In contrast to the first two cases, virtually no abnormalities in microtubular configuration were seen. In fact only two cilia showed any microtubular abnormality, and this was in the form of either a 7 + 2 configuration or a 9 + 2 + 1 configuration, in which a single extra microtubule was present within the confines of the outer ring of nine doublets. This frequency of tubular abnormalities was similar to that observed in the normal control patient in
41
IMMOTILE-CILIA SYNDROME
4
N '
s
FIG. 5. Cross section of five cilia from patient GJ. Note that the central microtubules are randomly oriented. I n n e r and outer dynein arms are absent, but nexin links (N), as well as other ciliary structures (spokes, central sheath), are clearly visible, x 151 000.
which 1.5%, of 154 cilia examined, were abnormal. The abnormal cilia were of the compound variety, i.e., two sets of 9 + 2 axonemal structures were present within the same cell membrane. DISCUSSION
The early studies of Gibbons (1965) and Satir (1965) have clearly shown that the inner and outer dynein arms correspond to an ATPase which is essential in generating an active sliding motion of one neighboring microtubular doublet relative to its neighbor. That the absence of one or both of the dynein arms results in partial or complete axonemal paralysis has been amply dem-
onstrated in humans (Afzelius et al., 1975; Afzelius, 1976; Afzelius and Eliasson, 1979), mice (Bryan, 1977; Bryan et al., 1977; Bryan and Chandler, 1978), and a paralyzed flagellar (pf) mutant of a Chlamydom o n a s strain (Huang et al., 1978). More recently, it has become apparent from experiments using a variety of pf mutants of C h l a m y d o m o n a s (Randall and Starling, 1972; Piperno et al., 1977; Adams et al., 1978; Witman et al., 1978), as well as from the examination of human material (Sturgess et al., 1979b, 1980; Afzelius and Eliasson, 1979), that the absence of other axonemal structures such as spoke heads and/ or spokes, one or both central microtubules, central microtubules, and central
42
S C H N E E B E R G E R ET AL.
sheath complex also results in flagellar paralysis. These observations support the studies of Warner and Satir (1974), who showed that components of the central sheath and/or spokes are essential for the conversion of interdoublet sliding into axonemal bending. The present study reports on several structural abnormalities in cilia from patients with the immotile-cilia syndrome which have thus far not been described, and demonstrates further that a large variety of structural abnormalities of respiratory cilia are associated with virtually identical clinical manifestations. The respiratory cilia of the first patient, JC, while lacking both dynein arms, were further abnormal in that 38% of the cilia contained one to nine supernumerary microtubules arranged external to the nine outer doublets. The effect that supernumerary microtubules might have on ciliary function is not known. It is conceivable that the presence of such extra microtubules, which are not connected to axonemal structures that are known to participate in the coordinated movement of cilia (Gibbons, 1976), might further impair ciliary function. Abnormalities in microtubular number and arrangement have been observed in patients with Kartagener's syndrome (Katz et al., 1977; Afzelius, 1977), chronic bronchitis (Ailsby and Ghadially, 1973; Konradova et al., 1975; McDowell et al., 1976), and lung carcinoma (McDowell et al., 1976). However, in none of these patients did the cilia with abnormal microtubular formations constitute more than 5% of the cilia examined. Of interest is a recent report in which as many as 24% of respiratory cilia of patients with retinitis pigmentosa were observed to have a variety of microtubular malformations (Arden and Fox, 1979). While the underlying mechanism responsible for the presence of supernumerary microtubules in such a large number of cilia in patient JC is unclear, it is probably not due to the chronic infections which are a prominent feature of the immotile-cilia syndrome. Being the young-
est patient, in the present series, JC had not experienced as many respiratory infections as the remaining two patients (GB and GJ) whose cilia did not have supernumerary microtubules. Furthermore, the cilia of GJ, which have the morphological characteristics of classical Kartagener's syndrome, were remarkably free of any microtubular abnormalities. The cilia of patient GB similarly demonstrated a number of unusual structural abnormalities. Forty-six percent of his respiratory cilia had the morphological features associated with a spoke defect; that is, eccentric central microtubules, and the displacement of one of the outer doublets toward the center. This finding differs from that originally described by Sturgess et al. (1979a) in which 100% of the cilia showed this shift in microtubular placement. Furthermore, in the three patients with the spoke defect which were examined by Sturgess and her colleagues, both dynein arms as well as nexin links were said to be present. In GB the inner dynein arms were absent and the outer ones were unusually prominent. Nexin links were not seen in any of the cilia from patient GB. Although nexin links may be difficult to resolve, they were readily seen in both of the other two patients (JC, G J) as well as in the normal control. Thus far no pf mutant of C h l a m y d o m o nas has been described in which there is an absence of nexin links. Indirect evidence gained from pf mutants which lack radial spokes, but in which nexin links are present (Witman et al., 1978), suggests that nexin finks form permanent bridges between adjacent outer doublets, and may, therefore, serve to maintain the structural integrity of the axoneme against dynein-generated shear forces. The present observations in patient GB are somewhat surprising in this regard, since more complete outer doublet disarray might be expected in cilia which lack nexin links and also have a spoke defect. It is conceivable that the outer dynein arms act, at least in part, to keep the outer
IMMOTILE-CILIA SYNDROME
doublets in place and are able to do so even in the absence of nexin links. While the purpose of the present study was to examine in detail the different structural abnormalities in three patients with the immotile-cilia syndrome, it is of interest to note that the father of GB had a moderate number (9% of cilia examined) of abnormal respiratory cilia, as well as some abnormal sperm. Furthermore, GB had a sibling who died at 9 months of congenital heart disease. Although Kartagener's syndrome, as defined by the clinical triad of situs inversus totalis, bronchiectasis, and sinusitis, is thought to be inherited as an autosomal recessive (Rott, 1979), the mode of inheritance of the various structural abnormalities of cilia which give rise to the immotile-cilia syndrome remains to be established. This work was supported by USPHS Grants HL25822, HL-07193, and HD-04807 and by Grant RR 128 from the General Clinical Research Centers Program of the Division of Research Resources of the National Institutes of Health. The examination of the sperm samples by Dr. Don Fawcett and the clinical coordination by Mrs. Pamela Hawley are gratefully acknowl-
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BRYAN, J. H. D., BATES, T. J., AND CHANDLER, D. B. (1977) Excerpta Med. Found. Int. Congr. Set. 426, 52. BRYAN, J. H. D., AND CHANDLER, O. B. (1978) J. Cell Biol. 79, 281a. ELIASSON, R., MOSSBERG, B., CAMNER, P., AND AFZELIUS, B. A. (1977) N. Engl. J. Med. 297, 1-6. GERALD, P. S., SCHUSTER, S. R., RUCKMAN, R. N., AND SCHNEEBERGER,E. E. (1978) Pediat. Res. 12, 515. GIBBONS, I. R. (1965) Arch. Biol. 76, 317-352. GIBBONS, I. R. (1976) in BRINKLEY, B. R., AND PORTER, K. R. (Eds.), International Cell Biology, pp. 348-357, Rockefeller Press, New York. HUANG, B., PrPERNO, G., AND LUCK, D. J. L. (1978) J. Cell Biol. 79, 286a. KARNOVSKV, M. J. (1965) J. Cell Biol. 27, 137. KARTAGENER,M. (1933) Beitr. Klin. Tuberk. 83, 489501. KATZ, S. M., DAMJANOV, I., CARVER, J., SPITZER, S., MASON, D., KAUFFMAN, L. A., AND SOLNICK, P. (1977) N. Engl. J. Med. 297, 1011-1012. KONRADOVA, V., HLOUSKOVA, Z., AND TOMANEK, A. (1975) Folia Morphol. 23, 293-295. McDowELL, E. M., BARRETT, L. A., HARRIS,C. C., AND TRUMP, B. F. (1976) Arch. Pathol. Lab. Med. 100, 429-436. PEDERSEN, H., AND MYGIND, N. (1976) Nature (London) 262, 494495. PEDERSEN, H., AND REBBE, H. (1975) Biol. Reprod. 12, 541-544. PIPERNO, G., HUANG, G., AND LUCK, D. J. L. (1977) Proc. Nat. Acad. Sci. USA 74, 1600-1604. RANDALL, J., AND STARLING, D. (1972) in BEATTY, R. A., AND GLUECKSOHN-WAELSCH,S. (Eds.), The Genetics of the Spermatozoon, pp. 13-36, University of Edinburgh, Edinburgh. ROTT, H. D. (1979) Hum. Genet. 46, 249-261. SATIR, P. (1965) J. Cell Biol. 26, 805-834. STURGESS, J. M., CHAO, J., AND TURNER, J. A. P, (1979b) J. Cell Biol. 83, 179a. STURGESS, J. M., CHAO, J., AND TURNER, J. A. P. (1980) N. Engl. J. Med. 303, 318. STURGESS, J. M., CHAO, J., WONG, J., ASPIN, N., AND TURNER, J. A. P. (1979a) N. Engl. J. Med. 300, 53-56. WARNER, F. D., AND SATIR, P. (1974) J. Cell Biol. 63, 35-63. WITMAN, G. B., PEUMMER, J., AND SANDER, G. (1978) J. Cell Biol. 76, 729-747.