Ultrastructural Features of Respiratory
Cilia in Cystic Fibrosis
SHEILA MORIBER KATZ, M.D., AND DOUGLAS S. HOLSCLAW, JR., M.D.
Katz, Sheila Moriber, and Holsclaw, Douglas S., Jr.: Ultrastructural features of respiratory cilia in cystic fibrosis. Am
J Clin Pathol 73: 682-685, 1980. The respiratory cilia of nine
patients with cystic fibrosis were examined by electron microscopy. In contrast to patients with "immotile cilia syndrome,"
the cilia from the patients with cysticfibrosiscontained dynein
arms and radial spokes. A low percentage of abnormal cilia
were detected in all nine patients, but, except for the occurrence of rippled cilia in these patients, the alterations were
similar both in morphologic terms and incidence to alterations
in a control group of patients with chronic bronchitis. Lesions
included compound cilia, excess cytoplasmic matrix, and an
abnormal number or arrangement of microtubular doublets.
Patients suffering from cystic fibrosis do not exhibit ultrastructural ciliary alterations characteristic of immotile cilia
syndrome. (Key words: Respiratory cilia; Cystic fibrosis;
Immotile cilia syndrome.)
PATIENTS with "immotile cilia syndrome" exhibit
sinusitis, nasal polyposis, chronic otitis media, chronic
bronchitis, bronchiectasis, recurrent infections of the
respiratory tract, impaired mucociliary clearance, and
immotile spermatozoa.'• 4 - 7 Some also have associated
situs inversus of Kartegener's syndrome. All patients
studied to date have absent mucociliary transport
and one patient displayed absent ciliary movement
in the middle e a r . 1 5 " Characteristic ultrastructural
alterations of both respiratory cilia and sperm flagella
consist of absence of dynein arms and abnormalities
in number and arrangement of microtubular doublets.
Cilia with defective radial spokes are a recently described morphologic variant of this syndrome."
Patients with cystic fibrosis also exhibit chronic
recurrent respiratory infections, sinusitis, nasal polyposis, bronchiectasis and infertility. Studies of mucociliary clearance in patients with cystic fibrosis suggest an abnormality in ciliary clearance of pulmonary
secretions. I2,13 The similarity of symptoms in patients
Received April 23, 1979: received revised manuscript and accepted
for publication September 14, 1979.
Supported in part by grants from the Cystic Fibrosis Foundation
and a Pediatric Pulmonary Center Grant from the Bureau of
Maternal and Child Health Services, Department of Health,
Education and Welfare.
Address reprint requests to Dr. Katz: Department of Pathology,
Hahnemann Medical College and Hospital, 230 North Broad Street,
Philadelphia, Pennsylvania 19102.
Departments of Pathology and Pediatrics, Hahnemann
Medical College and Hospital, Philadelphia,
Pennsylvania
with cystic fibrosis and those with the immotile cilia
syndrome prompted our investigation of the ultrastructure of cilia of patients with cystic fibrosis.
Methods
Patients
Cystic Fibrosis (Group I). Cystic fibrosis was diagnosed in nine patients by elevated values of sweat
electrolytes obtained by the pilocarpine iontophoresis
method (quantitative) and a typical clinical history,
sputum culture and radiographic findings. The patients'
age range was seven to 28 years with a mean age of
20 years. There were five male patients and four female
patients. Of the three men who were tested, all were
aspermic.
Spoke
Dynein
arm
Microtubular doublet
FIG. 1. A diagram of a normal cilium illustrates nine peripheral
microtubular doublets and two central microtubules. The peripheral
doublets are connected to each other by nexin links (not shown)
and the central microtubules by radial spokes. Dynein arms containing adenosine triphosphatase are attached to one of each of the
outer microtubular doublets.
0002-9173/80/0500/0682 $00.70 © American Society of Clinical Pathologists
682
BRIEF SCIENTIFIC REPORTS
Vol. 73 • No. 5
683
Table I. Alterations of Respiratory Cilia in Cystic Fibrosis
Abnormal
Number or
Age
(Years)
Group I
Patient
1
Source
Total
Abnormal
Cilia (%)
Compound
Cilia
Cytoplasmic
Excess
Matrix
Arrangement
of Microtubular
Doublets
Presence
of Dynein
Arms
Presence
of Radial
Spokes
Other
Alterations
28
Nasal
turbinate
Patient
2
26
Nasal
turbinate
Patient
3
27
Nasal
turbinate
Patient
4
19
Bronchus
Patient
5
25
Nasal
turbinate
Patient
6
28
Nasal
turbinate
Patient
7
7
Nasal
polyp
Patient
16
Nasal
polyp
Cytoplasmic protrusions, rippled
contour [5r/<)
Patient
9
10
Nasal
polyp
Cytoplasmic
protrusions
Group II
Patient
1
Rippled contour (89?)
Megacilia. rippled
contour (8Cf)
Small cilia
Trachea
Patient
2
6
Bronchus
Patient
3
13
Bronchus
Patient
4
23
Nasal
turbinate
Patient
5
13
Nasal
turbinate
Megacilia, cytoplasmic protrusions
Cytoplasmic protrusions
* This patient was four months old.
Chronic Bronchitis (Group II). Five patients with
chronic bronchitis or bronchiectasis served as a control
group. Sweat tests were normal, and immune competence was intact. The age range was four months to
23 years with a mean age of 11 years. There were four
male patients and one female patient. The one male
tested had normal numbers and motility of sperm.
Tissue
Biopsy of bronchus, trachea, posterior aspect of
the middle nasal turbinate, nasal polypectomy and
pneumonectomy provided cilia for ultrastructural
examination. All operative procedures were for diagnostic or therapeutic purposes. Informed consent was
obtained from each patient.
A dissecting microscope was used to identify the
surface bearing cilia. Each sample was fixed in 2.5%
glutaraldehyde, postfixed in 1% osmium tetroxide,
dehydrated with increasing percentages of alcohol,
embedded in Spurr® and sectioned for routine electron
microscopy. For each case, a minimum of 500 cilia
were examined, and the percentage of abnormal cilia
was calculated per specimen.
KATZ AND HOLSCLAW
684
*
»• \ , '
;
;
A.J.C.P. • May 1980
•
V «
(it) >*
?2a
'
2b
%,<&
^ *5*
• f;
.V
y
2c
FIG. 4. One microtubule is missing (arrow) from the central region
of this cilium. Uranyl acetate and lead citrate, x 150,000.
FIG. 2. Four deformed cilia are illustrated: o, b, compound cilia;
c, a cilium exhibiting excess cytoplasmic matrix: d, a deformed cilium containing an abnormal number of microtubular
doublets and a rippled external contour. Uranyl acetate and lead
citrate, a: x50,000, b: x22,000,c: x52,000rf: x52,000.
Results
Group 1
The cilia in each specimen contained dynein arms
and radial spokes. Most of the cilia were normal
(Fig. 1). The incidence of abnormal cilia ranged from
2 to 7%, except for rippled cilia (Table 1). Ciliary alterations were observed in all patients and are summarized
in Table 1. All patients exhibited some compound cilia
(Figs. 2a and 2b); seven patients had excessive cytoplasmic matrix (Fig. 2c); three patients had rippled
cilia (Fig. 3); seven patients had an abnormal number or
arrangement of microtubular doublets (Figs. 2d and 4).
Megacilia, small cilia and cytoplasmic protrusions were
noted in some patients (Table 1).
Group II
^
> . ' * • «
- * . .
a
» . «
K ^ * ; j" ^J^$&«
V"
., X V
' /Irs****? v*^\
%
^
Mil!
ttWFIG. 3. A cluster of cilia, featuring rippled external contour,
were obtained from Patient 4 (group I). The cilia contain radial
spokes and dynein arms. Uranyl acetate and lead citrate, x60,000.
The total incidence of ciliary alterations and the
types of alterations observed were comparable to those
of group 1 (Table I), except there were no rippled cilia.
Discussion
In contrast to patients with immotile cilia syndrome,
cilia from patients with cystic fibrosis displayed dynein
arms and radial spokes. The observed alterations of
cilia consisted principally of compound cilia, excessive
cytoplasmic matrix, abnormal number or arrangement
of microtubular doublets and rippled ciliary contour.
These ultrastructural lesions were present in both
nasal and bronchial epithelial cilia. The occurrence of
similar ciliary alterations in our control group and in
previously described inflammatory bronchial lesions2-™-""'" suggests that our observed cilial deformities
are secondary to inflammation of the respiratory tract.
Although the percentage of altered cilia in the present
study was low, it is conceivable that the lesions detected might impede mucociliary clearance and contribute to the progressive pulmonary pathologic condition characteristic of cystic fibrosis.
BRIEF SCIENTIFIC REPORTS
Vol. 73 • No. 5
Acknowledgments. John A. Tucker, M.D., Professor and Chairman of the Department of Otolaryngology helped obtain the
specimens, and Ms. Andrea Polin provided technical assistance.
7.
8.
References
9.
1. Afzelius BA: A human syndrome caused by immotile cilia.
Science 193:317-319, 1976
2. Afzelius BA: Ultrastructure of cilia and flagella, Handbook of
Molecular Cytology. Edited by A Lima de Faria. Amsterdam,
North Holland Publishing Co., 1969, pp 1219
2. Ailsby RL, Ghadially FN: Atypical cilia in human bronchial
mucosa. J Pathol 109:75-78, 1973
4. Eliasson R, Mossberg B, Camner P, et al: The immotilecilia syndrome. New Engl J Med 297:1-6, 1977
5. Fischer TJ, McAdams JA, Entis GN, et al: Middle ear ciliary
defect in Kartagener's syndrome. Pediatrics 62:443-445,
1978
6. Howell JT, Schochet SS Jr, Goldman AS: Ultrastructural defects
10.
11.
12.
13.
685
of respiratory tract cilia associated with chronic infections.
Arch Pathol Lab Med 104:52-55, 1980
Katz SM, Damjanov I, Carver J. et al: Kartagener's syndrome
and abnormal cilia. New Engl J Med, 297:1011-1012, 1977
Kondradova V, Hlouskova Z, Tomanek A: Atypical cilia in
human epithelium from large bronchus. Folia Morphol
(Praha) 23:293, 1975
McDowell EM, Barrett LA, Harris CC. et al: Abnormal cilia
in human bronchial epithelium. Arch Pathol Lab Med
100:429-436. 1976
Neustein HB, Church J, Cohen S: Dysmorphology of cilia.
JAMA 241:2423, 1979
Sturgess JM, Chao J, Wong J, et al: Cilia with defective radial
spokes. New Engl J Med 300:53-56. 1979
Wong JW, Keens TG, Wannamaker EM, et al: Effects of
gravity on tracheal mucus transport rates in normal subjects
and in patients with cystic fibrosis. Pediatrics 60:146-152.
1977
Yeates DB, Sturgess JM, Kahn SR, et al: Mucociliary transport
in trachea of patients with cystic fibrosis. Arch Dis Child
51:28-33, 1976
Myospherulosis —Further Observations
THOMAS M. WHEELER, M.D., AND MALCOLM H. McGAVRAN, M.D.
Wheeler, Thomas M., and McGavran, Malcolm H.: Myo-
spherulosis—further observations. Am J Clin Pathol 73:
685-686, 1980. Myospherulosis has recently been shown to be
composed of altered erythrocytes. Mysopherules have been
produced in vivo and in vitro using a petrolatum-based tetracycline antibiotic ointment (Achromycin®). In the reported
study, myospherules were produced in vitro using either lanolin
or petrolatum, the two components of the vehicle of this ointment. Additionally, human fat produced myospherules in vitro.
(Key words: Myospherulosis; Erythrocytes.)
MYOSPHERULOSIS is an accumulation in tissue of
sac-like structures enclosing smaller endobodies. Since
its description in 1969 by McClatchie and Bremner.5-6
and after that by Hutt, 3 mysopherulosis has evoked
considerable discussion as to the nature of the saccules
and endobodies, which resemble an endosporulating
fungus. Rosai has recently shown that myospherules
are derived from erythrocytes. 7 This was demonstrated
in vitro using fresh, packed human erythrocytes and
a widely used tetracycline antibiotic ointment (Achromycin®). We have extended this research and shown
that both lanolin and petrolatum, components of the
vehicle of this ointment, produce myospherules in
Received May 7, 1979; received revised manuscript and accepted
for publication July 10, 1979.
Address reprint requests to Dr. Wheeler: Department of Pathology, Baylor College of Medicine, Houston, Texas 77030.
Department of Pathology, Baylor College of Medicine
and the Methodist Hospital, Houston, Texas
vitro. That two unrelated lipids mixed with erythrocytes produced myospherules led us to try human fat,
and this also produced mysopherules.
Materials and Methods
One milliliter of fresh human erythrocytes was added
to each of a series of test tubes containing approximately 1 ml lanolin, petrolatum, a petrolatum-lanolin
mixture, (9:1 petrolatum-lanolin, the approximate
proportion of these substances in tetracycline ointment), and emulsified human fat that had been disrupted from its cellular confines with mortar and pestle.
The negative control was a test tube containing erythrocytes only; the positive control was a test tube containing 1 ml tetracycline ointment to which was added
1 ml of erythrocytes. The contents of each test tube
were mixed thoroughly for 30 sec with a separate
wooden applicator stick. The tubes were placed in an
incubator at 37 C for 24 hours. After removal from
the incubator, each specimen was fixed overnight by
70% ethanol. Each specimen was then processed into
paraffin, cut into 5-/xm sections, and stained with
hematoxylin and eosin.
0002-9173/80/0500/0685 $00.60 © American Society of Clinical Pathologists