nasal polyps. patients with asthma, aspirin ...

Ultrastructural and functional studies of cilia from patients with asthma, aspirin intolerance, and nasal polyps. F H Lewis, T F Beals, T E Carey, S R Baker and K P Mathews Chest 1983;83;487-490 DOI 10.1378/chest.83.3.487 The online version of this article, along with updated information and services can be found online on the World Wide Web at: http://chestjournal.chestpubs.org/content/83/3/487

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Ultrastructural and Functional Studies of Cilia from Patients with Asthma, Aspirin Intolerance, and Nasal Polyps* F H. Lewis,

M.D.;t

T F Beats,

S. R. Baker,

M.D.;II

and

M.D.;

T E. Carey,

K. P. Mathews,

Ph.D.;

M.D.11

asthmatics often have sinus disease, nasal polyps, and obstructive airways disease. This presentation is reminiscent of the symptoms of persons afflicted with various forms of ciiary dyskinesia. Therefore, we examined tissues from seven “triad” asthmatic patients for ciiary structural or functional abnormalities. Transmission and scanning electron microscopy revealed no specific abnormalities. Cultures of nasal epithelia were maintained for up to 20 “Triad”

ecently, much attention structural abnormalities tion

with

respiratory

has been ofcilia

disease,”7

tagener’s to as the immotile cilia have identified motion

infertility,2’8

Although syndrome,2 in some

tion

described syndromes and the immotile

to the

originally

more recently have included of microtubules,3’7 tubules

with

transposition

central

area.4

Although

ciliary

bronchial

tion,

these

disturbed meninges

lack

ofdynein

abnormal first

also

ciliary

arms,9

abnormalities in the inversus

Afzelius’4

five

from

associated

addi-

polyps and obstructive

had spirometric airways disease.

the

Departments

Otorhinolaryngology, the Ann Arbor Arbor, MI. tFellow in Allergy.

male

cases,

tAssociate

Professor

Veterans

and

two

aspirin

connection

was

with

one

and

nasal

surgery biopsies

were inferior

and/or

cocaine

as a topical

readings which suggested In addition, chronic sinus

diately

placed

phosphate

Administration

Medical

Pathology School,

Center,

and

the

and Ann

pieces.

the

then scopy.

of Pathology.

§Assistant Research Scientist in Otorhinolaryngology. Associate Professor of Otorhinolaryngology. #{182}Professor of Internal Medicine. Manuscript received June 21; revision accepted October 19. Reprint requests: Dr Mathews, Department oflnternal Medicine, University ofMichigan Medical Center, Ann Arbor 48109

motility

with

The and

and were

were

scanning

and

examined

15 percent

fetal

acids,

p.g/mi

amphotericin

calf

100 U/mi

changed

weekly,

on bits

of tissue

serum,

tests

removed

of ciiiary from

with

penicillin,

2 mm

100

washing, X 2 mm

gluteraidehyde

micro-

for initial

ciliary

supplemented

glutamine,

nonessential

streptomycin,

activity

were

and

0.02

Medium made

to hemocytometer

CHEST/83/3/March,1983

and

electron

medium

atmosphere.


imme-

washed

B. After

CO,

culture

were

then

microscopy

100 pg/mI

B in a 5 percent and

and

transmission

2 mM

penicillin,

Nasal of the

of 4 percent

100 U/mi

37#{176}Cin Eagle’s

situs

undergo-

included.

specimens

2 percent

by light

at

adults

approximately into

then cultured

amino

into

biopsy

areas

amphotericin

placed

in was

with

an application

biopsy

containing

p.g/ml

a nasal subject

were

salt solution

divided were

for both rest

.02

sample

control,

also

after

(PBS)

a semen

normal-appearing

The

balanced

saline

pieces

processed

on

In three

challenge

from

for from

documented

polyps.

careful

male

specimens

turbinates

streptomycin, Some

addition,

reasons

anesthetic.

buffered

by

as a positive

unrelated

(protocol Grants Beings)

well

nasal

an 18-year-old

performed

in Earles

specimens

In

Two control

for

punch

and

confirmed

and from

sinusitus.

Human with

asthma,

patient,

consent, to Review

nonsmokers

study.

obtained

informed

Involving

adult

another

middle

Medical

female

was

the

Medicine,

on

subjects.

Committee

Investigation

intolerance

nasal

developed

of Michigan

and

also have since this

undertaken

control

with

intolerance,

with

inversus

obtained,

and

of aspirin

from

of Internal University

Research

histories

pg/ml

*From

were

and

by the institutional

specimen

patients

were

approved

was

obtained

that

studies

asthmatics

biopsies

with

frequently

polyps

be expected to aggravate the disclearance occurring at the beginepisodes . ‘ Accordingly, ultrastruc-

functional

of “triad”

middle ear, sinuses, and in halfofaftlicted human

noted

syndrome

findings

nasal

METHODS

ing

cilia

and

cilia

these In

are

often

of asthmatic

tural

These

with

(“triad” asthma) might abnormality, particularly

abnormality would turbed mucociliary ning

problem.

asthmatics

a rather unique subpopulation of asthmathave severe disease. If present, such an

to be

ics who

Nasal

14

immotile

seems

Clinical

clearance

and

a prominent

to us that

aspirin intolerance some type ofciliary

to the

in sperm,

mucus

was

suggested

other

of micro-

microtubules

described

affect

drainage

abnormalities ectopic position

numbers

trachea2’4 function and situs

embryos In 1979,

“

ofouter

abnormalities

the

dyskinesis. Sleigh’2 ciliary dyskinesia” encompass the pre-

ultrastructural radial spokes,3

and

Kar-

including Kartagener’s cilia syndrome. In addi-

noted

reported defective

and

generally referred other observers,4” of the cilia and sug-

gested the use ofthe term “ciliary has noted that at present, “primary is the preferred term. This would viously syndrome

focused on ultraand their associa-

weeks. They showed normal ciliary activity which was not influenced by perfusing the tissue with medium containing aspirin. In contrast, control tissue from a patient with situs inversus and sinusitis showed the expected structural and functional ciliary abnormalities. In culture, the ciliary function of tissue from this patient could be partly restored by perfusion with ATP or ATPase.

was

periodically chambers

487

/\

1. Far left, Diagram ofcross section ofcilium showing normal axoneme arrangement of nine outer microtubule doublets with inside and outside dynein arms and two central microtubules. Center left, Cilium from “triad” asthmatic patient with normal appearance. Center right, Two cilia from patient with situs inversus and chronic sinusitis: one cilium with a 9 + 2 with translocation ofa peripheral microtubular doublet and one cilium with a 9 + 0 with translocation ofa peripheral microtubular doublet arrangement. Far right, Cross sectional cilium from normal control patient with abnormal dynein arms (arrows) (original magnification x 110,000). FIGURE

perfused with reagents dissolved 370 C. These bits oftissue were

one

and

nine

additional placed

trial into

days

after

in Earle’s balanced assessed for ciliary

being

was performed

with

Ciliary

culture.

to 3 in a manner

similar

placed cilia

motility to that

into

tissue

removed

visually

by

after

jects

and

being of 0

et al’6

obtained

nasal

seven

patients

biopsy

with

specimens

“triad”

showing

asthma, ciliated

five

had

epithelium.

One of the male patients with a nasal only squamous metaplasia provided sample. An additional patient had

biopsy revealing us with a semen squamous meta-

plasia

with

of the

biopsied

nasal

mucosa,

rare

ciliated

only by light microscopy. Scanning and electron microscopy of those specimens

with

epithelium

obtained

asthmatics all showed ultrastructurally Fig 1 (far left) is a diagramatic cross As shown

in Fig

peripheral

1 (center

doublets

left),

and

dynein

their normal relationships. “triad” asthmatic patient the

nasal

about

cilia

from

46 percent

patient with two types

number

these

section

normal cilia. ofa cilium.

central

arms

“triad”

pair,

were

the

control

of the

cilia

situs inversus of defects.

subjects. from

and Some

of microtubules

the

control

showed one microtubular

central pair placement

and with

1 [center

of

others a nor-

right]).

favorably oriented cross sections of cilia, dynein were visible on nearly all peripheral doublets. had shortened arms, visible on a doublet.

in

the one as were

positive

In arms Some

and rarely the arms were not No cilia were seen with totally

absent dynein arms. Individual shortened or, apparently absent dynein arms were seen in occasional cilia from our normal controls (Fig 1 [far right]), and so the significance of this finding is uncertain. Light and phase contrast microscopy of the nasal biopsy 488

specimen

from

the

five

“triad”

asthmatic

with

were cilia

one

specimens good

of the

from

the patient stifl

“triad”

minimal

motion

with

bristle-like

situs cilia

observed

to-side

pattern

with

The

pieces

of ciliated

revealed

wave-like

motile. This finding seen in the other

revealed

bacteria culture

Sperm

patients,

as noted

is consistent patients. In

with the contrast,

inversus

and

little

occurred

little

normal-

motion.

with

sinusitis

motion.

in a slow,

or no bending tissue

and/or fungi. Sections for 1#{189} months were

toluidine

The

stiff side-

of the

remained

blue.

sub-

These

oftissue stained

cilia.

viable

sections

which with

revealed with little indicating

the ciliated

necrosis

“triad”

epithelium Small

asthmatics

specimens

in

aspirin minutes

culture

with

underlyall of the the

larger

2 pg/ml for 30 ciliary

with 200 pg/ml aspirin solution for produced no observable change. for 30 minutes with a solution

a combination acid 2.66 x 10

activity.

from from

perfused

of the

to no uptake viability of ofthe

tissue

periodically were

were in aqueous

in phosphate buffered saline any perceptible change in

motility; perfusion 60 minutes also Likewise, perfusion

ciliary

ofciliated

taken

solution without

containing arachidonic

despite

pieces

in were by

uptake

stain by the ciliated epithelium in the underlying lamina propria, ing tissue.

In contrast,

sinusitis showed

(Fig

nine

present

Sperm tails from likewise were normal

transposition with absent revealed ectopic microtubular mal

from

the

control

tissue culture for as long as 20 weeks. Cultures eventually discarded because of contamination

cells identified transmission ciliated

two

function from

above, normal tissue

RESULTS

Of the

the

appearing An

on a scale

Forrest

at

culture.

112 days

was scored employed

salt solution activity between

Perfusion

the ciliary activity patients undergoing

with

of aspirin M yielded aspirin

of the control nasal surgery

2 p.g/ml no change

and in

also did not affect tissue from for unrelated

two rea-

sons. In view

of the

report

by Forrest

ATPase can enhance the activity with the immotile cilia syndrome, from our patient with situs inversus perfused with 5 x 10” glml ATP ATPase. Enhanced ciliary motion three and five different experiments, Ultrastructural

and Functional

Studies

et al’6 that of cilia from nasal tissue

Patients

and

patients cultures

and sinusitis or 2.38 x 10 was observed respectively,

of Cilia from Asthmatic


ATP

were glml in with

(Lewis et a!)

SITUS INVERSUS

-

CHRONIC SINUSITIS

FIGURE

2. Ciliary

administration CILIARY MOTION

Earle’s 37 C

ATPase

tient

ATP

20

40

60

80

100

120

140

TIME

either versed

agent. The by washing

enhanced out the

anced

salt

and

solution

ciliary cultures

could

160

IN

180

motion

of the

“triad”

asthmatics

and

controls.

Afzelius’7 has suggested that the ultrastructural abnormalities seen in cilia can be separated into two groups: those with the specific defects he associates the

immotile

as nonspecific. we have seen subjects category. abnormalities

would

cilia

syndrome

The occasional in our “triad” all be

Indeed, noted

and

those

morphologic asthmatics

classified

under

regarded

his

rest

ditionally, function

aspirin of the cilia

did not from the

assessed.

have triad

any effect asthmatics

.

Adon the We also

used arachidonic acid in the perfusate along with aspirin, since by blocking the cycbooxygenase pathway ofarachidonic acid metabolism, aspirin might cause a shift to the lipoxygenase pathway with subsequent production of leukotrienes . The latter are believed to be important mediators of allergic and inflammatory reactions,’8 but no change in the ciliary motility resulted. These experiments do not exclude the pos-

that approximately any apparent This seen

one motility

in the to 37#{176}

factor to that

half of his cilia did in vitro on initial

does not explain, in the beating ofthe

since

ofthe

the

however, rest ofthe

ultrastructural

cilia appeared

in vitro. in cilia

the cilia

Rossman which

normal.

In addition

et al’9 have

appeared

in

appearance it is ultrato lack

demonstrated

immobile

through

the

use of a video motion analyzer attached to a phase contrast microscope. If one postulates that the cilia without apparent motion were the ones demonstrating

nub-like with an

been

a serum similar

whether the cilia with the abnormal were the same ones that appeared

appears that cilia from patients with the triad of aspirin sensitivity, nasal polyposis, and asthma do not differ significantly from normal in the ultrastructural and have

to

The electron micrographs of his that approximately 46 percent in appearance correlated with the

syndrome’9),

ultrastructural

which

ciliary

(the stift slow, brush-like motion previously described above and noted as a type of abnormal motion seen

fuzzy. Afzelius noted that when dynein arms were seen in his patients with Kartagener’s syndrome, they were shorter than those found in normal cilia. Others also have noted these shortened arms.5’6 In any case, it

parameters

.

similar

fibrosis.

examination. abnormalities

motion

or

Median

.

in a manner

or the increase in temperature chamber from room temperature C.

300

ciliary dyskinesia.4 cilia demonstrating were abnormal

motility in

inversus.

scored

aspirin might interact with a ciliary inhibitory factor

in cystic

unknown structure

nonspecific

situs

was

Our patient with chronic sinusitis and situs inversus had findings compatible with the diagnosis of primary

ofthe

even most of the ultrastructural in our patient with situs inversus

and sinusitis were of this nonspecific type, but addition, the dynein arms present on the microtubule doublets in this case occasionally were nub-like

functional

280

sibility that to produce

this

changes normal

and

260

observation not evidence

DISCUSSION

with

240

seen

by reperfu-

sion with ATP or ATPase (Fig 2). Ofnote was the finding that not every cilium observed increased its motion with the perfusion of these solutions: some cilia (less than 30 percent) remained immotile. The ATP and ATPase had no definitive effect on the already active ciliary

220

MINUTES

motion was rewith Earle’s bal-

be restored

200

to sequential in pa-

ATPase

Forrest et The initial, small enhancement in ciliary motion after simply perfusing with Earle’s solution may just reflect slight mechanical stimulation from the medium perfusing the chamber andl

x

0

with

. .

motility

-6

response and

of AlT

defects,

then

one

would

have

to suggest

that other factors (eg, biochemical) might be involved in the abnormal motion evidenced by the ultrastructurally normal looking cilia. It has been suggested that

outer least

dyneim altered

arms are only partial physiologic response;

dynein arms in mollusks).

only on this microtubules

of dynein complete

consist ofthree or four subunits However, we found these nubs

patient’s of both

triad patients. ATPase has been

pieces the

microtubules, normal control found

but also on subjects and

to be a prominent

(at not the the

structural

component ofdynein arms, and ATP is a major source ofenergy for ciliary movement.2’ The latter appears to involve an interaction between dynein arms and the adjacent peripheral doublet.2’ In view ofthese considerations, Forrest et aP6 tried stimulating ciliary activity ofpatients with the immotile cilia syndrome with ATP CHEST


I 83

I 3 I

March,

1983

489

and

ATPase

ciliary

and

activity.

with

the

cilia

sinusitis,

reported We, of our

the

Although

with

situs

inversus

effect

of ATP

surprising

that

might

as large

ex-

the

ciliary

cell.

In

activity

case,

the

for prolonged

tory epithelial ongoing work human

any

periods

cultures provides on the effect ofvarious

ciliary

an

11

opportunity relevant agents

caused

by

for on

immotile

factor in chronic Med 1977;297:1-6 3 Sturgess

JM,

J Med

Engi 4 Sturgess

P. Afzelius

ciliary

BA.

The

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and

cilia.

J,

J,

Wong

spokes:

Another

J

N EngI

a cause

Aspin

N,

of human

Turner

JAP

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respiratory

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with

disease.

cause

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ciliary

ofciliary

motility.

N

Cilia

D, and

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19

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StJ,

bronchiectasis. Neustein

with

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Steele

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in Polynesian

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of Cilia from Asthmatic


Patients

(Lewis

et a!)

Ultrastructural and functional studies of cilia from patients with asthma, aspirin intolerance, and nasal polyps. F H Lewis, T F Beals, T E Carey, S R Baker and K P Mathews Chest 1983;83; 487-490 DOI 10.1378/chest.83.3.487 This information is current as of July 12, 2011 Updated Information & Services Updated Information and services can be found at: http://chestjournal.chestpubs.org/content/83/3/487 Cited Bys This article has been cited by 1 HighWire-hosted articles: http://chestjournal.chestpubs.org/content/83/3/487#related-urls Permissions & Licensing Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://www.chestpubs.org/site/misc/reprints.xhtml Reprints Information about ordering reprints can be found online: http://www.chestpubs.org/site/misc/reprints.xhtml Citation Alerts Receive free e-mail alerts when new articles cite this article. To sign up, select the "Services" link to the right of the online article. Images in PowerPoint format Figures that appear in CHEST articles can be downloaded for teaching purposes in PowerPoint slide format. See any online figure for directions.
