Effect of Inhalation of Single Dose of Beclomethasone on Airway Epithelium

Effect of Inhalation of Single Dose ofBeclomethasone on Airway Epithelium

Jirı Uhlık, MD and

Ludek Vajner, DVM, PhD

Department of Histology and

Embryology, Charles University,

2nd Faculty of Medicine, Prague,

Czech Republic

Jana Ad�aa�sskov�aa, MS

Institute of Computer Science,

Academy of Sciences of the

Czech Republic, Prague,

Czech Republic

V�aaclava Konr�aadov�aa, MD, PhD

Department of Histology and

Embryology, Charles University,

2nd Faculty of Medicine, Prague,

Czech Republic

ABSTRACT Inhaled corticosteroids are being recommended for the treat-

ment of bronchial asthma for their anti-inflammatory properties and

reduction of airway hyperreactivity. The first tissue coming to the contact

with all inhaled substances is the airway epithelium. In this experiment,

the immediate effect of a single MDI dose of beclomethasone on the ultra-

structure of the tracheal and bronchiolar epithelium was studied. Due to the

beclomethasone administration, the secretory elements were highly affec-

ted. The tracheal goblet cells were damaged, mucus release was significantly

accelerated, and the mechanism of secretion was influenced. The bronchio-

lar Clara cells revealed signs of the pathological alteration. Their secretory

granules were usually stored in the cytoplasm. Occasionally, degenerating

Clara cells were found after the beclomethasone administration. The injury

of ciliated cells in both locations was only mild and this fact was reflected in

slight impairment of the tracheal ciliary border. As a morphological sign of

impaired self-cleaning ability, inspissated secretion was discovered among

cilia. According to this evaluation, the inhalation of the single dose of beclo-

methasone caused a moderate damage to the tracheal epithelium and a mild

one to the epithelium of terminal bronchioles. The results draw attention to

the adverse effects of otherwise therapeutically beneficial inhaled glucocor-

ticosteroids.

KEYWORDS electron microscopy, glucocorticosteroids, rabbit, terminal

bronchioles, trachea

Glucocorticosteroids (GCS) are used as effective components of the

first-line therapy in patients suffering from the mild to severe persistent

bronchial asthma [1]. They are being recommended for their anti-

inflammatory properties and reduction of the airway hyperreactivity [2, 3].

The effects of GCS action are mediated either by a direct binding of GCS=

glucocorticosteroid receptor (GR) complexes to the special sites in the

promotor regions of responsive genes, or by an interaction of this com-

plexes with transcription factors such as activating protein-1 and nuclear

factor-jB [4].

The most widely used route of the GCS administration in patients suffer-

ing from bronchial asthma is inhalation that allows increasing the local drug

Received 18 January 2007; accepted27 March 2007.

The authors thank professor PetrPohunek, head of the respiratorydepartment at the Paediatric Clinic,University Hospital Motol, Prague, forhelpful criticism of the manuscript.The experimental work wassupported by research project No.111300003 of the Ministry ofEducation of the Czech Republic andgrant No. 79=97 of the Grant Agencyof the Charles University, Prague,Czech Republic.

Address correspondence to Jirı Uhlık,MD, Department of Histology andEmbryology, Charles University, 2ndFaculty of Medicine, V Uvalu 84,CZ-150 06, Prague 5 – Motol, CzechRepublic. E-mail:[email protected]

Ultrastructural Pathology, 31:221–232, 2007Copyright # Informa HealthcareISSN: 0191-3123 print=1521-0758 onlineDOI: 10.1080/01913120701425951

221

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concentration with minimizing systemic bioavailabil-

ity of GCS [5]. The inhaled GCS can be administered

either as an aerosol by metered-dose inhalers (MDI),

spacers, and nebulizers, or as a finely milled powder

by dry-powder inhalers (DPI) [6, 7]. The first tissue

coming in contact with all inhaled substances is the

airway epithelium. Both a and b GR isoforms were

proved in airway epithelial cells [8]. Nevertheless,

the direct effect of GCS drugs on this epithelium in

vivo has not been studied in detail.

From the inhaled GCS family, beclomethasone

dipropionate (BDP) was chosen in our experiments.

BDP is a topically active GCS used in the treatment

of asthma and rhinitis since 1972. BDP is actually a

pro-drug with a weak GR binding affinity that is

hydrolyzed via esterase enzymes to the active meta-

bolite beclomethasone 17-monopropionate [9]. Anti-

inflammatory and anti-hyperresponsive potency of

inhaled BDP is approximately twice lower than in

newer inhaled GCS fluticasone propionate, thus its

dosage must be higher [10, 11]. Nevertheless, this

drug remains the most frequently used inhaled GCS

in clinical practice.

We decided to study the morphological reaction of

the airway epithelium to the administration of BDP

on the ultrastructural level. To be able to compare

our results to the previous ones, we concentrated to

the immediate reaction of the airway epithelial cells

to the single dose of BDP. As experimental models,

rabbits were used because their airway epithelium

best resembles the human epithelium among all com-

mon laboratory animals [12].

MATERIALS AND METHODS

In our experiments, 15 specific pathogen-free New

Zealand White rabbits (body weight 1500–3000 g,

Charles River, Sulzfeld, Germany) were used. All

experimental procedures were performed under gen-

eral anesthesia, using the IM administration of a mix-

ture of ketamine (35 mg=kg bw, Narkamon, Spofa,

Prague, Czech Republic) and xylazine (5 mg=kg bw,

Rometar, Spofa, Prague, Czech Republic). The

experiment was certified by the Animals Protection

Expert Committee of the 2nd Faculty of Medicine,

Charles University, Prague, Czech Republic.

Three animals were treated with 2 MDI puffs of

Becotide Inhaler (Glaxo, Greenford, United

Kingdom; efficient substance – BDP, propellants –

trichlorofluoromethane, dichlorodifluoromethane,

oleic acid). In one puff, 50mg of BDP was contained.

Single dose administered to each rabbit represented

100 mg. The remaining 12 rabbits represented 2 con-

trol groups. Six animals (treated controls) were admi-

nistered 2 MDI puffs of Placebo Inhaler (Glaxo,

Greenford, United Kingdom) containing only pro-

pellants. Six remaining rabbits represented the

untreated group and were bred under the same con-

ditions as all previous animals. The unequal numbers

of animals in experimental groups were caused by

technical reasons as we simultaneously performed

light-microscopic histochemical examinations in 2

control groups (results not published). Three

animals in both control groups were used for the

electron-microscopic evaluation of the tracheal

mucosa and the other 3 for the evaluation of terminal

bronchioles.

The MDI nozzle was connected with a plastic IV

16-G catheter (outer diameter 1.7 mm, inner diameter

1.3 mm, length 6.5 cm) that was inserted shortly into

the mouth of a rabbit, and 2 puffs of aerosol were

administered during spontaneous inspirium. The

canister was actuated in an inverted vertical position.

During the whole procedure, the experimental

animals breathed quietly under the general anesthe-

sia. The untreated control animals were only

anesthetized.

Material for electron microscopic examination was

collected 30 min postexposure. This interval was

chosen pursuant to our previous studies, in which

30 min was approved as a time sufficient for the dis-

tinct reaction of the epithelium [13, 14]. Still under

the general anesthesia, the ventral cervical region

of the animals was additionally infiltrated subcuta-

neously by procaine (Procain, Leciva, Prague, Czech

Republic) 10 min before preparation. The rabbits

were sacrificed by removal of their lungs and hearts.

Tiny fragments of tracheal membrane

and=or lungs were collected and processed using

standard methods for electron microscopy. The

material was fixed for 90 min with 5% glutaralde-

hyde (Merck, Hohenbrunn bei Munchen, Germany)

in cacodylate buffer (pH 7.2) and then for 60 min

with 2% OsO4 (JMC, Hertfordshire, United Kingdom)

in cacodylate buffer (pH 7.4), dehydrated in graded

series of alcohol and embedded in a Durcupan–

Epon mixture (Fluka, Buchs, Switzerland). Ultrathin

J. Uhlık et al. 222

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sections were prepared on Ultrotome Nova (LKB,

Broma, Sweden), contrasted with uranyl acetate

and lead citrate and examined under the JEM 100 C

electron microscope (Jeol, Tokyo, Japan).

In the tracheal epithelium, the functional state of

the goblet cells and the ciliary border were evaluated

quantitatively. The goblet cells were identified and

classified directly on the fluorescent screen of the

electron microscope. Only the cells showing their

apical surfaces were included. The number of ana-

lyzed samples and fields per animal depended on

the number of goblet cells found, as these cells are

distributed unequally in the epithelium. We evalu-

ated from 50 to 223 goblet cells per animal. To evalu-

ate the distribution of goblet cells in the epithelium,

the isolated elements and the goblet cells arranged in

groups were distinguished. The secretory elements

were further classified into 3 categories: (1) nonsti-

mulating, (2) mucus-discharging, (3) degenerated.

Kinocilia were counted using transparent grids in

electronmicrographs displaying transverse sections

of the ciliary border and their average numbers per

1mm2 of the ciliary border were estimated. In indi-

vidual rabbits, we evaluated the areas from 263.75

to 1,145,25mm2. Four categories of kinocilia were

distinguished in the same electronmicrographs: (1)

intact 9þ 2 cilia, (2) slightly damaged pathological

cilia with local swellings of the ciliary membrane or

with tiny vacuoles situated in their shafts, (3) degen-

erating cilia, represented by axonemes incorporated

into cytoplasmic blebs or by isolated axonemes, and

(4) malformed cilia with either abnormal arrange-

ment or number of microtubules in their axonemes.

In the specimens of transversely sectioned ter-

minal bronchioles, the total number of ciliated and

Clara cells and the functional state of Clara cells

(presence or absence of secretory granules) were

recorded on the fluorescent screen of the electron

microscope. Again, only the cells showing the apical

surfaces were included. The number of evaluated

cells (116–336 per animal) depended on the number

of terminal bronchioles found in our material. At

least 2 samples were observed in each animal. Using

the computer image analyzer Lucia G (Laboratory

Imaging, Prague, Czech Republic), details of Clara

cells’ cytoplasm were evaluated in randomly selected

digitalized electronmicrographs of their supranuclear

portions. The supranuclear cytoplasm was marked

and measured. In this area (754–1507mm2 per

animal), secretory granules and mitochondria were

delineated and counted and their areas were auto-

matically gauged.

For statistical evaluation, relative values of individ-

ual categories of tracheal goblet cells, bronchiolar

epithelial cells, Clara cells, and cilia were evaluated

by the v2 test of homogeneity in frequency tables.

To specify categories causing deviations from the

hypothesis of homogeneity, adjusted standardized

deviations were used. Means of cilia=mm2 were com-

pared by the one-way analysis of variance (ANOVA).

The differences between groups were assessed by

the Tukey’s test or Bonferroni’s method for multiple

comparisons. Levene’s test for equal variances was

also performed. As a nonparametric analogy of the

ANOVA, the Kruskal-Wallis test was used. The results

of the computer image analysis were statistically

evaluated by the ANOVA. The differences between

individual groups were tested by the two-sample

t test (software NCSS version 6.0).

RESULTS

The tracheae of all rabbits were lined with the

pseudostratified columnar epithelium composed of

ciliated, goblet, and basal cells. Isolated cells of the

diffuse neuroendocrine system (DNES) and a few

differentiating ciliated or secretory elements were

also encountered. Intercellular spaces were narrow

and apical junctional complexes remained intact.

The epithelium rested on a well-developed basal

lamina.

In animals treated with both MDI inhalers (Pla-

cebo Inhaler and Becotide Inhaler), the reaction of

ciliated cells was mild (Figure 1). Only a few tiny

cytoplasmic protrusions exceptionally containing

isolated axonemes of degenerating kinocilia were

found on their apical portions. A slight increase in

number of small vesicles and lysosomes and an

inconspicuous dilatation of cisternae of rough endo-

plasmic reticulum and of Golgi complex were

observed in deeper portions of the cytoplasm. Mito-

chondria of some ciliated cells were slightly altered

(Figure 1b). Exceptionally, larger intracytoplasmic

ciliated vacuoles were revealed.

Goblet cells were mostly scattered as isolated

elements among the ciliated ones. In untreated

and treated controls, only 6� 3% and 8� 1%

(mean� SD) of them formed tiny groups, mostly

223 Beclomethasone and Airway Epithelium

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FIGURE 1 Apical portions of tracheal ciliated cells, bars 0.5 mm. (a) Intact cytoplasm of a ciliated cell and a well-developed apical

junctional complex, untreated control. Original magnification, 20,0003. (b) Small vesicles and slightly altered mitochondria in the cyto-

plasm of a ciliated cell, treated control. Original magnification, 20,0003. (c) Small vesicles and intact mitochondria in the cytoplasm of

two ciliated cells and a well-developed apical junctional complex, BDP. Original magnification, 20,0003.


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composed of 2 cells. After the administration of BDP,

15� 2% of goblet cells were arranged in groups

In both control groups, the goblet cells were

mostly nonstimulated and filled with mucus. In

untreated controls, only 3� 1% of them discharged

their mucus from apical granules (Figure 3a). Degen-

erated goblet cells were not found at all. In treated

controls, 16� 1% of the goblet cells were stimulated

to discharge mucus (12� 1% mucus-discharging

and 4� 0% degenerated) (Figure 2). Mucus was

evacuated from apical mucus granules. Only excep-

tionally, packets of mucus granules were detached

or signs of compound exocytosis were noticed in sti-

mulated goblet cells (Figure 3b).

After the BDP administration, the secretory ele-

ments were highly affected: 71� 2% of the goblet

cells were stimulated to discharge mucus (Figure 2).

The secretion was liberated from apical

granules, but also signs of an apocrine type of

secretion and of a rapid compound exocytosis were

noticed in numerous cells. Voluminous packets of

mucus granules were detached from apical portions

of some stimulated goblet cells. Chain fusion of

adjacent mucus granules’ membranes followed by

instantaneous evacuation of the whole mucus load

was also recorded. 58� 4% of goblet cells were

completely exhausted and revealed signs of

degeneration. After rapid release of mucus, the

exhausted cells did not take part in further secretory

cycles but degenerated (Figure 3c). They gradually

lost their connections with the basal lamina and

were sloughed off. Remnants of their condensed,

degenerated cytoplasm were found in the area of

the ciliary border.

Regular ciliary border was developed above the

tracheal epithelium of all animals (Figure 4). In

untreated controls, treated controls, and BDP-admi-

nistered animals, the average number of cilia per

1 mm2 was 9.7� 0.3, 8.4� 0.4, and 8.8� 0.5, respect-

ively. Kinocilia were mostly intact; altered kinocilia

represented only 1.2� 0.1%, 4.4� 1.0%, and

2.4� 0.6%, respectively (Figure 5). After the BDP

administration, clumps of inspissated secretion and

whole layers of condensed mucus were observed

in the area of the ciliary border (Figure 4c).

The terminal bronchioles of all rabbits were lined

with a simple epithelium where low columnar or

cuboidal ciliated cells and high columnar Clara cells

alternated almost regularly. In the epithelium of

untreated controls, treated controls, and BDP-

administered animals, the ciliated cells represented

47� 4%, 44� 1%, and 47� 2% of epithelial cells,

respectively. The percentage of the Clara cells was

53� 4%, 56� 1%, and 53� 2% (Table 1). The cells

rested on a well-developed basal lamina, apical junc-

tional complexes were intact, intercellular spaces

remained narrow.

Similarly as in the tracheal epithelium, the ciliated

cells revealed mild marks of pathological alteration

in both treated groups. On their apical surfaces with

tufts of cilia, formation of small cytoplasmic protru-

sions was exceptionally ascertained. The apical blebs

interfered with the regular arrangement of cilia, but

usually did not incorporate them. Increase in num-

bers of heterogeneous lysosomes and multivesicular

bodies were observed in deeper portions of the cyto-

plasm. Cisternae of Golgi complex were often

dilated. After the BDP administration, perinuclear

cisternae were dilated in several ciliated cells.

In treated controls, only about half of the secretory

Clara cells were intact. Their apical cytoplasm was

rich in thin tubules of smooth endoplasmic reticulum

filled with a moderately electron-dense content, large

mitochondria and electron-dense secretory granules.

Portions of small Golgi complex were also observed.

Exceptionally, larger granules filled with fibrogranu-

lar content similar to mucus ones were observed.

The perinuclear and basal portions of these cells con-

tained narrow cisternae of rough endoplasmic reticu-

lum arranged in parallel pattern. Remaining Clara

FIGURE 2 Quantitative evaluation of functional state of goblet

cells (GC) in tracheal epithelium of rabbits 30 min after inhalation

of 2 puffs of placebo inhaler and Becotide inhaler. N ¼ 3. Values

are expressed as means� SD. �Values significantly differ

(p < .01) from untreated control values. Values connected by lines

significantly differ (p < .01) from each other.


representing small intraepithelial mucus glands.

mucus

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FIGURE 3 Apical portions of tracheal goblet cells, bars 0.5 lm. (a) Mucus-filled goblet cell; mucus is evacuated from an apical mucous

granule, untreated control. Original magnification, 15,0003. (b) Fusing mucous granules in the apical portion of a stimulated goblet cell,

treated control. Original magnification, 15,0003. (c) Remnants of condensed cytoplasm of a degenerated goblet cell left in the epithelium

after evacuation of mucus, BDP. Original magnification, 15,0003.


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cells revealed signs of the pathological alteration.

They contained wider tubules of smooth endoplas-

mic reticulum filled with electron-lucent content,

cisternae of rough endoplasmic reticulum were

dilated, and mitochondria were altered (Figure 6b).

The pathological changes were more pronounced

FIGURE 4 Tracheal ciliary borders, bars 0.5 lm. (a) Intact regular ciliary border, untreated control. Original magnification, 20,0003.

(b) Regular ciliary border containing numerous pathological cilia (arrowheads), treated control. Original magnification, 20,0003. (c) Layer

of inspissated mucus embedding free kinocilia in the ciliary border, BDP. Original magnification, 20,0003.


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in some cells, but degenerated Clara cells were not

revealed.

After the BDP administration, most Clara cells

revealed no signs of alteration. Some Clara cells

contained dilated spaces of smooth endoplasmic reti-

culum and Golgi complex. Occasionally, degenerating

Clara cells with vacuolized cytoplasm, altered

mitochondria, and damaged nuclei were found in

the epithelium (Figure 6c). Remnants of detached

degenerated cells were recorded in the lumen of

terminal bronchioles.

Secretory granules were found in the majority of

Clara cells (74� 9%, 82� 12%, and 80� 11%).

The granules were usually stored in the cytoplasm.

Morphological signs of evacuation of their content

were found only exceptionally. The results of the

computer image analysis of the Clara cell supranuc-

lear cytoplasm are given in the Table 1.

DISCUSSION AND SUMMARY

The direct effects of GCS on the airway epithelial

cells have been studied mostly in vitro, and authors

have come to ambiguous conclusions. Dorscheid

and his co-workers found damage and apoptosis of

cultured airway epithelial cells exposed to 4 GCS,

including BDP [15, 16]. Conversely, Wen and his

group described inhibition of lung epithelial cell

apoptosis after treatment of cell cultures by dexa-

methasone [17]. Similar results were observed after

the budesonide treatment [18].

Experimental studies in animal models dealing

with the effect of GCS on the airway epithelium

have been rare. Pavlovic et al. described reduced

number and height of tracheal epithelial cells in rats

exposed to intramuscular injections of triamcinolone

for 7 days [19]. Dorscheid’s in vitro results were

supported by an in vivo study in mice treated with

intraperitoneally injected dexamethasone for 2–4

weeks. The authors described increased epithelial

shedding and apoptosis both in small and large

airways [20].

We started our study of the effect of GCS on the

ultrastructure of the airway epithelium by the evalu-

ation of the immediate response to the single inha-

lation dose. To the experimental animals, the drug

was administered in a dose proposed for small

children [11]. Standard chlorofluorocarbon BDP

MDI devices produce particles of approximately

3.5–4 mm in diameter (mass median aerodynamic

diameter, MMAD) [21]. A catheter extending the can-

ister nozzle modifies the particle size. In the study of

Hess and his co-workers, a 19-G catheter was used

FIGURE 5 Altered kinocilia in the tracheal ciliary border of rab-

bits 30 min after inhalation of 2 puffs of Placebo Inhaler and Beco-

tide Inhaler. N ¼ 3. Values are expressed as means� SD. yValue

significantly differs (p < .05) from untreated control value; �values

significantly differ (p < .01) from untreated control values; values

connected by lines significantly differ (p < .01) from each other.

TABLE 1 Quantitative Evaluation of the Epithelium of Terminal Bronchioles and Cytoplasm of Clara Cells (CC) in Rabbits 30 min after

Inhalation of 2 Puffs of Placebo Inhaler and Becotide Inhaler

Untreated

controls

Placebo

Inhaler

Becotide

Inhaler

Ciliated cells (%) 47� 4 44� 1 47� 2

Clara cells (%) 53� 4 56� 1 53� 2

CC with granules (%) 74� 9 �82� 12 �80� 11

CC without granules (%) 26� 9 �18� 12 �20� 11

Granules per 1 mm2 of CC cytoplasm 0.17� 0.07 0.17� 0.05 0.16� 0.02

Area of granules in CC cytoplasm (mm2) 0.15� 0.04 0.13� 0.01 0.10� 0.02

Mitochondria per 1 mm2 of CC cytoplasm 0.73� 0.35 0.77� 0.09 �1.19� 0.08

Area of mitochondria in CC cytoplasm (mm2) 0.22� 0.08 0.16� 0.01 �0.11� 0.02

Note. N ¼ 3. Values are expressed as means� SD, values designated � differ significantly (p < 0.05) from untreated controls, values connected by a linediffer significantly (p < 0.05) from each other.


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and the MMAD of BDP particles was 3.14� 0.61mm

for 10-cm length [22]. Another study described

salbutamol particles delivered by 19-SWG catheter

with the MMAS 2.0� 0.1mm for 22-cm length [23].

Our catheter was slightly thicker and shorter; never-

theless, we suppose that the aerosol particles

delivered by it were within the respirable range of

1–5mm [23]. Studies with monodisperse bronchodila-

tor aerosols showed that the therapeutically optimal

particle size was approximately 3mm. Larger particles

(5–6mm) were mostly deposited in oropharynx and

smaller particles (1.5 mm) reached preferentially

alveolar regions [24, 25].

In our experiment, the target cells for the GCS

exposure were mostly the tracheal goblet cells. The

proportion of stimulated goblet cells differed signifi-

cantly (p < .01) compared with the findings both in

the group of untreated rabbits and also in the animals

treated with propellants. Mucus release was signifi-

cantly accelerated and the mechanism of secretion

was influenced. In healthy untreated rabbits, only

3� 1% of secretory elements gradually discharged

propellant administration, the acceleration of mucus

secretion was only mild. Mucus was mostly evacu-

ated only from individual apical granules; the detach-

ment of groups of mucus granules and massive

formation of communications between neighbouring

mucus granules were encountered only exception-

ally. After administration of BDP, signs of an apoc-

rine type of secretion and of a rapid compound

exocytosis were noticed frequently. Whole packets

of mucus granules were detached from apical por-

tions of some stimulated goblet cells. Also chain

fusion of adjacent mucus granules’ membranes fol-

lowed by instantaneous evacuation of the whole

mucus load, which has been interpreted as the most

escalated mode of mucus cell secretion, was

encountered [26, 27]. After rapid mucus discharge,

the overstimulated goblet cells mostly degenerated

and were gradually sloughed off. Remnants of their

condensed, highly electron-dense cytoplasm were

frequently observed among free kinocilia. Compared

with both untreated and treated controls, the

proportion of the degenerated goblet cells differed

significantly (p < .01).

We have demonstrated that high level of stimu-

lation of goblet cells in the tracheal epithelium

accompanied with degeneration of about 50% of

these secretory elements induced a massive differen-

tiation of new secretory cells [27, 28]. As the differen-

tiating goblet cells retained the ability to divide [29],

the result of this process was hyperplasia of secretory

elements with changes in their distribution in the

epithelium [27, 28]. After administration of BDP, the

onset of differentiation of new secretory elements

demonstrated by changes in the goblet cell distri-

bution was noticed. The number of goblet cells

arranged in groups was twofold compared with that

found in both control groups (p < .01).

The ultrastructural changes of bronchiolar

secretory cells were not so prominent, but they cor-

responded with our findings described in the epi-

thelium of terminal bronchioles of rabbits exposed

to various experimental procedures [30, 31]. The

most prominent were the signs of Clara cell

pathological alteration both after administration of

propellants and BDP, but degenerative changes with

the detachment of degenerated Clara cells to the

lumen of terminal bronchioles were observed only

in rabbits treated with BDP. The significantly higher

(p < .05) proportion of Clara cells containing

secretory granules denoted the initiative stage of

their stimulation to produce secretion in both treated

controls and animals treated with BDP. After the BDP

administration, the computer image analysis of the

Clara cell supranuclear cytoplasm showed an

increase in relative number and a decrease in aver-

age size of mitochondria (p < .05), but not granules.

This finding can be explained by the more promi-

nent pathological alteration of the cytoplasm.

In both airway levels, the degree of injury to the

ciliated cells was mild and did not differ from that

produced in these elements by propellant adminis-

tration. In comparison with untreated control rab-

bits, only mild, but significant (p < .05) decrease in

average number of kinocilia in the tracheal ciliary

border was noticed both in treated controls and after

BDP administration. In these groups, also the rela-

tive number of altered kinocilia increased

(p < .01). Among the altered elements, the slightly

damaged pathological cilia prevailed. Due to the

inhalation of propellants, the proportion of altered

kinocilia was almost twofold compared with group

administered by BDP (p < .01). We hypothesized

that after BDP administration, the great amount of

mucus, discharged all at once from the mucus-

secreting elements, embedded and also protected


mucus from individual apical mucus granules. After

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FIGURE 6 Apical portions of bronchiolar Clara cells, bars 0.5 lm. (a) Narrow tubules of smooth endoplasmic reticulum, intact mitochon-

dria and secretory granules in the cytoplasm of an intact Clara cell, untreated control. Original magnification, 10,0003. (b) Dilated spaces

of smooth and rough endoplasmic reticulum and altered mitochondria in the cytoplasm of a Clara cell, treated control. Original magnifi-

cation, 15,0003. (c) Altered mitochondria, dilated tubules of smooth endoplasmic reticulum and a damaged nucleus of a degenerating

Clara cell, BDP. Original magnification, 15,0003.


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free kinocilia from the contact with injurious sub-

stances.

After the BDP inhalation, clumps of inspissated

secretion and whole layers of condensed mucus

were observed in the area of the ciliary border. These

findings represent morphological signs of impaired

self-cleaning ability of the tracheal epithelium [32, 33].

Propellant inhalation did not induce these changes.

The accumulation and condensation of mucus after

BDP inhalation was apparently caused by enormous

amount of mucus evacuated from more than 70% of

secretory cells in the tracheal epithelium.

Based on our previous experiments, we proposed

a classification of the degree of injury to the tracheal

epithelium [33]. To estimate the damage of the epi-

thelium we considered the percentage of stimulated

goblet cells, the degree of acceleration of their

secretion, the average number of kinocilia=mm2, the

percentage of altered kinocilia, and the appearance

of the morphological signs of the impairment of

the self-cleaning ability of the epithelium. According

to this evaluation, inhalation of 2 puffs of BDP aero-

sol caused moderate injury to the tracheal epi-

thelium, while after the same amount of aerosol

containing only propellants, mostly only mild dam-

age of this epithelium was revealed (Table 2). The

effect of the same dose of BDP and propellants on

the epithelium of terminal bronchioles was less pro-

nounced. Clara cells, which secrete less voluminous

secretion rich in proteins and play other roles in air-

way biology [34], revealed many more discreet

changes than tracheal mucus-producing goblet cells.

We can only speculate about the mechanism by

which BDP caused the airway secretory cell stimu-

lation and epithelial injury. One hypothesis can be

based on the study describing induction of b2 adre-

nergic receptor function in human nasal mucosa

after the BDP treatment [35]. The effect of stimulation

of epithelial cells possessing activated adrenergic

receptors by natural catecholamines can be similar

to the artificial stimulation of the naive airway

epithelium by IV administration of epinephrine

described in our two previous studies [31, 36].

Our results try to draw attention of clinicians to the

fact that the epithelium of the respiratory passages

reacts at least on the ultrastructural level to any arti-

ficial provocations including simple MDI inhalation

of placebo. The addition of BDP into inhaled spray

significantly intensified the reaction. Thus, the clini-

cal use and dosage of all inhaled drugs, including

the otherwise therapeutically beneficial inhaled

GCS, should be reasonably controlled, considering

their potential adverse effects.

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and Becotide Inhaler

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controls

Degree of damagePlacebo

Inhaler

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RatioDischarging GC

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Effect of Inhalation of Single Dose of Beclomethasone on Airway Epithelium

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