Chrysotile asbestos exposures can produce in

Eur Respir J
1990, 3, 81-90
Chrysotile asbestos exposures can produce
an alveolitis with limited fibrosing activity
in a subset of high fibre retainer sheep
R. Begin, A. Cantin, P. Sebastian
Chrysotile asbestos exposures can provide an alveolitis with limited fibrosing
activity in a subset of high fibre retainer sheep. R. Begin, A . Cantin. P. Sebastien.
ABSTRACT: Inhalation of flbrogenlc mineral dust may enhance flbronectin
(Fn) production by alveolar macrophages and Increase flbrobla:st growth
activity (FGA) In lung lavage fluid. To investigate the relationship of these
changes to flbre retention and the development of asbestosis, we exposed
15 sheep to 100 mg Canadian chrysotUe flbres In 100 ml saline, and ten
sheep to 100 ml saline only, at ten day Intervals. The animals were studied
at 3 month Intervals. At month 18, ten sheep bad abnormal chest radiograph (category ~1) (group B) and five bad normal radiograph (category
0) (group A). Pulmonary function data indicated restrictive patterns or
abnormalities In both groups, more severe In group B. Sheep In group A
bad broncboalveolar lavage (BAL) cellularlty and biochemistry compa·
rable to controls; sheep In group B bad significant Increases In total BAL
cells (x2), macrophages (x2), neutrophlls ( x4) and eoslnophUs (x3), Increased BAL lactate dehydrogenase and Fn, but FGA and procollagen 3
comparable to controls. Fibre retention was significantly increased in an
exposed sheep and 2.5 x higher in group B vs group A despite similar
exposures (70 intratracheal, 100 mg chrysotUe Infusions). Enhanced fibre
retention in group B preceded the appearance of disease. This study
confirms our earlier observation linking Individual susceptlbiUty to development of alveolltls to individual dust burden and provides evidence that
the excess or fibre retention can be obsen•ed before detectable disease. In
addition, we report a chrysotlle-lnduced early alveolltls with Incompletely
expressed flbroslng activity at the time of initial radiographic detection.
The Intensity or the alveolltls Is related to the degree of fibre retention.
Eur Respir J., 1990, 3, 81-90.
Individual susceptibility of humans to development of
disease is well recognized. In humans chronically exposed to asbestos dust inhalation at work, it is well
documented that, for a given level of exposure, only a
fraction of the workers develop asbestosis [1-6]. To
investigate the so-called "individual susceptibility factor", immunological background, lung structure and lung
clearance capacity have been considered.
Reports on human immunological histocompatibility
in asbestos workers, including our own recent work
[7, 8], have failed to identify definite markers for susceptible individuals. Anatomical structure characteristics of
the major airways have been related to risk factors in
adverse pulmonary response to asbestos exposure [2],
and these characteristics may influence mucociliary clearance of the asbestos deposited in the lung.
Alveolar dust clearance in humans has not been directly studied as a determinant factor for individual
susceptibility to asbestosis. However, lung tissue fibre
burden has been found to be increased in asbestos
Unit~ de Recherche Pulmonaire, Universit~ de
Sherbroolce, Sherbroolce, Qu~bec, Canada and Dust
Disease Unit, McGill School of Occupational Health,
Montreal, Quebec, Canada.
Correspondence: R. B~gin, CHUS, Sherbrooke,
Quebec, Canada JJ H 5N4.
Keywords: Asbestos; chrysotile; pulmonary fibrosis.
Received: August 1988.
Accepted after revision September 1, 1989.
Supported by MRC Canada and lRSST Quebec,
Canada.
workers compared to the general population [9]. Asbestos workers with isolated airway disease have twice the
lung fibre content of workers without the airway disease
[10] but only 50% of the fibre content of patients with
asbestosis [11-14]. Analyses of bronchoalveolar lavage
(BAL) fibre content of workers with asbestosis were also
found to be significantly higher than values found in
exposed workers without asbestosis [15, 16].
Since exposure levels cannot be precisely determined
in human studies, it is not possible to compare groups of
workers with similar exposure but different disease activity. Furthermore, the currently available techniques for
measurement of fibre deposition and clearance using
radioactive fibres evaluate primarily the rapid phase of
dust clearance and its validity in reflecting the late slower
phase of dust clearance, or overall clearance, have not
been established.
Alternatively, fibre retention in the bronchoalveolar
space, as determined by BAL, has been documented in
humans [17, 18] and in our sheep model [19] to reflect
82
R. BEGIN, A. CANTIN, P. SEBASTIEN
lung tissue retention. Thus, with this approach we
estimated lung tissue fibre retention and related it to other
phenomena in the sheep exposed to chrysotile dusts.
Our sheep model of asbestosis, which utilizes multiple
intratracheal injections of the chrysotile dust as a means
of exposure, has reproduced the heterogeneity of
lung tissue response previously observed in humans [1-6]
and other animal models [20, 21]. This model made it
possible to establish that susceptible sheep were
retaining more asbestos fibres [22, 23]. The levels of
dust retention in our initial studies were measured only
after disease was established.
The present experiment was therefore designed to test
the above hypothesis of a dust retention related
individual susceptibility. Thus, we obtained repeated
measurements of BAL fibre content during the course of
induction of experimental asbestosis.
An additional objective of this study was to characterize the fibrogenic activity of the early chrysotile-induced
alveolitis in the susceptible sheep, at time of initial
radiograph detection, and to relate the individual response
of lung tissue to the de!:,>Tee of retention of the chrysotile
fibres.
Methods
Animals. Twenty five sheep weighing 25-40 kg were
used in this study. They were prepared and accustomed
to the pulmonary techniques as previously reported
[22, 23].
Experimental design. The flock was divided into a group
of ten sheep exposed to phosphate buffered saline (PBS)
only and a group of 15 sheep exposed to 100 mg UICC
Canadian chrysotile asbestos fibres in lOO m! PBS every
ten days. These fibres were relativity unifonn and well
characterized, 92% being less than 0.25 J.!m in diameter
and 20 J.!m in length. Exposures were carried out after
nasotracheal intubation via repeated slow infusions of
the suspension in the trachea at ten day intervals throughout the 24 month study.
Sequence of analyses. The animals were studied prior to
exposure and at three month intervals thereafter by chest
radiographs (CR), pulmonary function tests (PFT) and
BAL analyses. Transbronchial lung biopsies were not
obtained as we have previously correlated the histopathology of the early radiographic changes in this model
[24-27].
Chest radiograph. Each sheep was positioned on a mobile
cart with a wooden board and a grid cassette under the
thorax. The X-ray source was placed at a 30° caudal
angle, 2 feet from the cassette. The intubated animal was
held at total lung capacity (TLC) using a giant syringe,
and radiographs were taken at exposure factors 80 kV,
20 mAs, and 0.02 s. Each radiograph was scored
according to the International Labor Organization (fLO)
classification of radiographic profusion of parenchyma!
opacities [28]. Th is class ification recognizes the
existence of a continuum of change, from no opacity to
the most advanced category. The scores were converted
to a linear scale of 0-10 (12 categories) as follows: ILO
grade 0/- (clearly nonnal) and grade 0/0 (nonnal after
close examination) = 0 on the linear scale; 0/1 = 1; 1/0
= 2; 1/1 = 3; 1/2 = 4; 2/1 = 5; 2/2 = 6; 2{3 = 7; 3/2 =
8; 3/3 = 9; 3/4 = 10. In the ILO classification, four
categories are defined on the basis of these same
profusion scores: category 0 =profusion scores 0/-, 0/0,
and 0/1: category 1= profusion scores 1/0, 1/1, and 1/2;
category 2 =profusion scores 2/1, 2/2, and 2/3; category
3 =profusion scores 3/2, 3/3, and 3/4. The radiographs
in category 0 are generally considered normal
whereas those in category 1 or above are definitely
abnonnal.
Pulmonary function tests. The methods used in PFT
assessment of the sheep have been published previously
[23-26]. Briefly, transpulmonary pressure was monitored
with a naso-oesophageal 7 ml balloon catheter and an
airway catheter connected to a Hewlett-Packard 270
differential pressure transducer (Hewlett-Packard,
Waltham, MA). Gas flow at the airway opening was
measured by connecting the cuffed endotracheal tube to
a Fleisch no. 2 pneumotachograph (Dyna-sciences, Blue
Bell, PA) attached to a flow integrator recorder system
and a Mink data processing system (Digital Equipment,
Montreal, Quebec), for on-line analysis and storing of
the data. Each PFT measurement was obtained after three
inspiratory capacity measurements at a constant volume;
TLC was defined as the lung volume at a transpulmonary pressure of +35±5 cmHp, and residual volume RV
was defined as the lung volume at a transpulmonary
pressure of -35±5 cmHp. The static expiratory lung
compliance (Ctst) was determined by multiple-step syringe deflation between TLC and functional residual
capacity (FRC). Diffusion capacity (DLCo) was obtained
by a passive rebreathing method using a gas mixture of
10% helium, 0.30% carbon monoxide, and 21% oxygen
in nitrogen, a Collins catherometer (Warren E . Collins,
Braintree, MA) and a Beckman infra-red carbon monoxide analyser (Beckman Instruments, Fullerton, CA). For
the DLco test, the animals were passively ventilated with
a 2.5 I syringe at a rate of 30 breaths·min·1 with a 1 1
tidal volume.
Bronchoalveolar lavage and cell analysis. The techniques
in BAL procedures and analyses have been described
previously [22, 23]. The BAL effluent was passed through
four layers of cheesecloth to remove mucus, and the cells
were pelletized by centrifugation. Cells were counted in
a haemocytometer, and cell viability was determined by
the trypan blue exclusion technique. Cytocentrifuge
smears served to identify the cellular populations recovered with the Wright-Giemsa stain.
BAL proteins and enzymes. In the supematant, albumin
was determined by the immunochemical method of
Kn.LJNGSWORTH and SAVORY [29), with a laser nephelometer (Behring LN modular system, Hoechst Behring,
Frankfurt, DDR) using specific antiserum raised in
ALVEOLITIS IN CHRYSOTILE ASBESTOS EXPOSURE
rabbits (Cappel Lab. Inc., Downington, PA). The activity
of lactate dehydrogenase (LDH), an indicator of cytoplasmic toxicity, was measured by spectrophotometric
methods [30].
BAL mLJtrix constituents. To assess interstitial lung matrix
changes we looked at the fibronectin accumulations in
BAL fluid. We also measured the production of
fibronectin by BAL cells in culture [31, 32]. To determined cellular production of fibronectin, BAL cells were
incubated without adherence step in 24 well culture plates
(Linbro Chemical Co., New Haven, Conn.) in Dulbecco's
modified Eagles medium (DMEM, Gibco Diagnostic
Laboratories, Grand Island, NY) containing 100 Umi·1
penicillin and 100 Jlg·ml·1 streptomycin, at a density of
2x1()6-ml·1, for 1 h at 37°C, 10% C02 • At the end of the
incubation period, the supernatants were centrifuged
(500xg, 10 min) and stored at -20°C before fibronectin
measurement. Fibronectin was determined by
direct enzyme-linked immunoadsorbent assay (ELISA)
technique adapted from RENNARD et al. [31]. Briefly, 96
well polystyrene plates (Costar, Cambridge, Mass.) were
coated with gelatin (500 Jlg per well) at 20°C for 12 h,
rinsed three times with PBS, and samples of BAL supernatant were placed in each well at 20°C for 2 h. The
plates were then washed and the concentration of
fibronectin was determined by using antisheep fibronectin
antibodies raised in rabbits and peroxidase conjugated
goat antirabbit immunoglobulin G (IgG) (Boehringer
Mannheim Biochemicals, Mannheim, DDR). Using the
ELISA technique, the alveolar macrophages are the only
BAL cells producing significant amounts of fibronectin
[32].
Fibroblast proliferation assay. Since activated alveolar
macrophages can release increased amounts of molecules
that are mitogenic for fibroblasts [33, 34]. we tested the
release of fibroblast growth factors from lung inflammatory cells. Lung inflammatory cells were suspended in
Dulbecco's modified Eagles medium (DMEM) containing 100 U.ml·1 penicillin and 100 Jlg·ml·1 streptomycin at
a density of 1()6 cells·mP and incubated at 37°C, 10%
C02, for 18 h. The adherence step was omilted here as
macrophages have been documented to be the sole BAL
cell source of growth factor for fibroblasts [35). The cells
were then centrifuged (500xg, 10 min) and the supematants were passed through a 0.22 J.!M Millex filter
(Millipore, Bedford, MA) before use in the fibroblast
proliferation assay. Diploid human fetal lung fibroblasts
(HFL-1 CCL 153, American Type Culture Collection,
Rockville, MD) (Bradley et al.) were grown to
confluence in DMEM, 10% calf serum (Bocknek,
Rexdale, Ontario), treated with 0.5 mg·ml·1 trypsin and
0.2 mg·ml·1 edetic acid (EDTA), and seeded in 24 mm
culture wells (Linbro Chemical Co., New Haven, Conn.)
at a density of 5xl0'1 cells per well in DMEM 0.4% calf
serum, 10% C02 , 37°C for four days. The cells were
then washed three times with PBS and incubated with
supematants from alveolar macrophage cultures diluted
1:2 in DMEM 0.4% calf serum at 37°C, 10% C02 for
three days. At the end of the incubation, the cells were
83
treated with trypsin/EDTA for 15 min and counted in an
electronic particle counter (Model ZM, Coulter
Electronics Inc., Hialeah, FLA). Each experiment included
a negative control consisting of fibroblasts incubated with
DMEM alone and a positive control consisting of DMEM
+ 10% calf serum. The results were expressed as the
percentage increase in fibroblast number over control
values.
Electron microscopy. The analytical transmission electron microscope was used to detect chrysotile fibres in
lung lavages. All samples were prepared in a "clean
room", using filtered chemicals and carefully cleaned
glassware. Thirteen ml were isolated from the third
effluent of lavage and allowed to react at room temperature with sodium hypochlorite, in order to digest the
biological material. An aliquot of the suspension,
corresponding to 5 ml of BAL, was filtered on a polycarbonate membrane filter 25 mm in diameter with a pore
size of 0.2 j..lm (Nucleopore Corp., Pleasanton, CA).
Particles collected at the upper surface of the membrane
were embedded in a carbon film and subsequently
transferred onto 200 mesh grids [15]. Several grid
openings were observed in the transmission mode at a
screen magnification xlO,OOO. Chrysotile fibres were identified by their morphological features and their elemental
composition as determined by energy-dispersive X-ray
spectrometry (PGT system IVR; Princeton Gamma Tech,
Princeton, NJ). The length of each individual fibre encountered was measured directly on the screen to the
nearest 0.2 Jlm; diameter was measured using an eyepiece graticule. Fibre count analyses were performed at
months 3, 15 and 24 of the study.
Statistical analysis. In the presentation of the results,
values of the data for each group of sheep are followed
by the standard error as an index of dispersion. The data
were evaluated by analysis of variance for experiments
having repeated measurements on the same subjects.
When a significant effect was detected, a Kruskal-Wallis
test was used to determine which group means were
significantly different (p<0.05) [36). Analyses of correlation of fibres in BAL to other parameters of disease
activity were performed with the Spearman correlation
rank test [36].
Results
Subsets of sheep. At month 12, the group of 15 sheep
exposed to chrysotile had significant changes in radiographic score, static lung compliance, vital capacity and
arterial oxygen tension (Pao2) compared to saline exposed sheep and these changes were accentuated
thereafter. Analyses of individual results of the 15 asbestos exposed sheep showed that these changes were the
effect of disease limited to ten of the 15 sheep. The chest
radiograph was abnormal in ten. Analysis of lung functions and BAL cellularity of individual sheep with reference to the 95% confidence interval of control sheep also
clearly separated the diseased subset.
R. B~GIN, A. CANTIN, P. S~BASTIEN
84
We therefore divided the group of chrysotile exposed
sheep into two subsets: a subset of five sheep with nonnal
chest radiograph (0/0 or 0/1 score) (subset A) and a subset
of ten sheep with abnonnal chest radiograph (1/0 or above
score) (subset B). In this model of asbestosis, histopathological changes associated with early changes in chest
radiograph have been reported in detail [25-27] and
briefly consist of a diffuse peribronchiolar macrophagic
alveolitis extending into the adjacent interstitium and
alveolar spaces.
two chrysotile exposed sheep. The fibre length also significantly changed in the two groups over time. In the
chrysotile exposed sheep with nonnal chest radiograph,
the mean fibre length·J.1).·1 of BAL was 4.3±1.5, 4 .6±1.7
and 15.1±1.2 at months 3, 15 and 24, respectively. In the
chrysotile exposed sheep with abnonnal chest radiograph,
the mean fibre length was 3.2±1.4, 5.2±1.3 and 10.1±1.9
~·J.tl· 1 at months 3, 15 and 24, respectively. In both
groups, the average fibre length was significantly longer
at month 24 only (p<0.05).
Bronchoalveo/ar exposure index. This index is a time
integral of fibre estimated to be retained in the bronchoalveolar space. It is expressed as number of
fibres·J.1).· 1 per month. Results are presented in table 1.
For the two groups of sheep, which received the same
exposures, the bronchoalveolar exposure indices were not
significantly different.
Lung functions. In figure 1, we present the time course
of changes in chest radiograph scores in each of the three
groups of sheep in parallel with selected lung functions.
It can be appreciated that, whereas lung functions of
diseased sheep are clearly distinct from control sheep,
the functions of chrysotile exposed sheep with nonnal
radiograph remained between the normal and the
Table 1. - Fibre exposure and retention
Control
sheep
Sheep with normal
radiograph
Sheep with abnormal
radiograph
Exposure
Bronchoalveolar
exposure index
fibres·~-tl" 1 per month
0
1601±590
1881±360
<0.2
51.6±3
34.6±19
10.5±2.9
84±2*
91.6±2*
25.5±2.5*
4.3±1.5
4.6±1.7
15.1±1.2
3.2±1.4
5.2±1.3
10.1±1.9
Retention
Fibre number fibres·~-ti-1
Month
3
15
24
<0.2
<0.2
Fibre length 1.1Month
3
15
24
<1
<1
<1
Bronchoalveolar exposure index is a time integral of fibre estimated to be retained in the
bronchoalveolar space (fibres·~-tl" 1 per month). Results are geometric meariS±l SI!. *: p<0.05
for sheep with abnormal radiograph vs sheep with normal radiograph. At month 24, mean
fibre length was longer in exposed sheep but the difference between the two groups of
exposed sheep was not significant.
Fibre retention. BAL fibre analyses were obtained at
months 3, 15 and 24 of the study (table 1). In the saline
exposed sheep, fibre counts were always <0.2
fibres·~·! and all were shorter than 1 J.L in length. In the
chrysotile exposed sheep with nonnal chest radiograph,
fibre counts were 51.6±3, 34 .6±19 and 10.5±2.9
fibres·J.Ll" 1 at months 3, 15 and 24, respectively. In
the chrysotile exposed sheep with abnonnal chest radiograph, fibre counts were 84.1±2, 91.6±2 and 25.5±2.5
fibres·J.Ll" 1 at months, 3, 15 and 24, respectively. The
ratio of fibre counts for normal/abnonnal ~as 0.61, 0.37
and 0.41 at months 3, 15 and 24, respectively. Differences between the two groups were significant at p<0.05
at all times. A few asbestos bodies were observed in the
radiographically abnonnal groups. Lung resistance (RL)
at last point of study, month 24, was 0.24±0.08 kPa·/·1·s
in controls, 0.33±0.07 in subset A and 0.52±0.09 in subset
B (p<0.05 for B vs controls). These changes in RL were
accompanied by significant reduction of effective lung
compliance at 54 and 45% controls in groups A and B,
respectively. These values in both exposed groups suggested a peribronchiolar process as previously described
in the sheep model [22-24].
The alveolitis. In figure 2, we present results of cellularicy of lung lavage and in figure 3, the biochemistry and
cell culture results. In the saline exposed sheep, there
was no significant change over time in all parameters
ALVEOLITIS IN CHRYSOTILE ASBESTOS EXPOSURE
12
Profusion of parenchyma!
opacities
score
•
9
500
•
*
Static lung
compliance
6
ml·cmH20'1
400
4
300
3
200
2
85
VC I
6
3
•
100
0
3
6
9
12
15
19
21
24
0
3
6
9
120
Pao 2
12
1!5
19 21
24
0
torr
40
30
eo
20
•
DLCO
3
6
9
12
15
18 21
•
0
18
21
.24
Control sheep
3
6
9
12
15
18 21
**
pcO.OS
pcO.OS
v• control
v• sheep
with
normal radiograph
24
Month
Fig. I. - Results of chest radiograph scoring and lung
DLco: carbon monoxide diffusion capacity.
f~mction
tests in the three groups of sheep. VC: vital capacity; Pao2: arterial oxygen tension;
Total cell 1 x10'1·mf·1
*
0
6.0
15
Asbe.tos exposed sheep
with normal radiograph
.A. Asbestos exposed sheep
wllh abnormal radiograph
10
24
12
0
Month
60
9
ml·mln·1·torr·1
40
0
6
Month
•
100
60
3
Month
Month
3
6
9
12
15
•
19 21 2 4
Month
Eoslnophll1 x10'1·ml·1
4.!5
0
16
3
6
9
12
H5
9
1.5
4
0
3
6
9
12
1!5
Month
18 21
24
0
3
6
0
3
6
9
12
1!5
18 21
9
12
115
Month
*
•
3.0
24
Month
Neutrophil 1 x 10·1·mf·1
1.2
•
18 21
12
24
Month
Fig. 2. - Time course of bronchoalveolar lavage cellularity in the three groups of sheep identified as in figure I.
19
21
24
86
2oo
R. BEGlN, A. CANTIN, P. SEBASTffiN
Albumin
aoo Flbronactln
o.o6o Procollagen 3/albumln ng·11g·1
600
0.045
*
400
0
3
6
9
12 15 19 21
0
24
3
6
3
6
9
15
18 21
24
12
15
Month
0
3
6
9
19 21
24
12
15
18 21
2-4
Month
20 Flbronectln production*
ng·10-4 calls per 24 h
LDH miU·ml·1
0
12
Month
Month
40
9
1oo Flbroblast growth activity
15
75
10
~
~50
5
25
,
0
3
6
9
12
15
Month
19 21
2-4
0
3
6
9
12
15
18
21
24
Month
Fig. 3. - Tune course of selected biochemical marlcers of disease process in lbe three groups of sheep identified as in figure 1. The upper lbree
panels and the bottom left panel are from bronchoalveolar lavage (BAL) fluid analyses. The bouom middle and right panels are derived from BAL
cell cultures as detailed in the Methods section. LDH: lactate dehydrogenase.
except for the increase in lymphocytes which has not
been observed in our prior studies of the sheep model
and remains otherwise totally unexplained. This was not
observed in another conl.rol group of sheep evaluated
during the same two year period (our silicosis study).
The increase in lymphocytes in this study also caused
total BAL cells to be s lightly raised over time in
conlrols.
In the chrysotile exposed sheep with normal chest radiograph, group A, values of cellularity were comparable
to saline exposed sheep without significant changes in
lymphocytes over time which remained at 10±2% of total
BAL cells.
In the chrysotile exposed sheep with abnormal chest
radiograph, group B, there were significant increases in
total BAL cells, macrophages, eosinophils and neutrophils at month 12 and thereafter, compared to controls
and group A. Furthermore, BAL albumin was slightly
but significantly increased 50-70% above conl.rols with
parallel increases in IgG and IgM (data not shown) and
more accentuated (300% of controls, month 18) increases
in BAL l ac tate dehydrogenase. Fibroncctin (Fn)
production by macrophages in culture remained below
detection level (<2 ng·l06 cells per 24 h) in controls
throughout the study. In group A, F n did not differ
from controls. Fn in group B was increased above
controls after month 12 and accumulated to values 300%
of controls in the BAL supernatant of the sheep in
group B. These changes occurred in the absence of
significant increase in BAL procollagen 3/albumin ratio
or fibroblast growth activity.
Fibre retention correlates. Analysis of correlation of fibre
retention to other parameters of disease activity documented a significant relationship between individual fibre
retention and radiographic disease (p<O.Ol), cytotoxicity
(LDH in BAL) (p<O.Ol), fibronectin production by alveolar macrophages (p<O.Ol), increased cellularity of
BAL (p<0.02) and loss of vital capacity (p<0.05). These
associations strengthen the concept that, for a given
chrysotile exposure, the intensity of disease process
directly relates to the individual fibre retention.
Discussion
The present study reproduced the heterogeneity of lung
tissue response previously observed in sheep chronically
exposed to chrysotile [22, 23], in asbestos workers [1--6]
and in other animal models of asbestosis [20, 21]. The
sequential analyses of BAL fibre content, an index of
alveolar dust retention [15, 17, 23, 37] documented the
following observations which have not previously been
reported. Firstly, given that exposure was continued
throughout the 24 months of the study at the same
exposure level (one injected dose per ten day interval),
both groups of chrysotile exposed animals had a gradual
decrease in alveolar dust retention which implies eiCher
ALYEOLlTIS IN CHRYSOTILE ASBESTOS EXPOSURE
an enhancement of the alveolar clearance up the tracheobronchial tree or a larger transfer to the interstitial
space. Secondly, fibre length significantly increased after month 15 in both groups of exposed sheep. The
observation of a gradual decrease may be particular to
the model and/or mode of exposure, but it also concurs
with observations by WAGNER et al. [20] on rat asbestosis, and RoWLAND et al. [38] and S~BASTIEN et al. [39] in
human asbestosis. The enrichment in long fibres has also
been documented [39]. This enhanced alveolar clearance
was observed in both groups of sheep with or without
disease. Thirdly, the animals with asbestosis not only
retained significantly more fibres but a significant excess
of fibre accumulation could be detected before any
abnormality of pulmonary radiograph, function or
bronchoalveolar lavage. This observation therefore documented that this high retention state preceded the development of the disease. A link to individual susceptibility
for disease development can, therefore, be firmly
considered.
Our study is also of interest as it further characterized
the early stage of asbestosis at the time of initial radiographic recognition of the disease process. Previous studies in humans (4, 40-42] and in several animal models,
including sheep [22-27, 43-52] have clearly documented
that lungs chronically exposed to asbestos or other mineral
dusts have an excessive early accumulation of macrophages in the lower respiratory tract.
This initial macrophage alveolitis may not necessarily
lead to interstitial lung fibrosis. Indeed, it has been
documented in long-term asbestos and silica workers [53]
that the macrophage population of the lower respiratory
tract may be expanded in the absence of interstitial lung
fibrosing disease; in the animal model, it is a wellrecognized early lung tissue reaction to inert dust
particles which usually disappears without residual scar
in the months after exposure cessation [54, 55]. This
type of "transient macrophagic alveolitis" has been
observed following latex bead, carbon, graphite, and
particulate carborundum exposures (54-56]. Pathological characteristics of this lesion are its absence of distortion of the normal histological lung structure and the
clearance of the phagocyte accumulation after cessation
of exposure [54-56).
In terms of cell biology of the macrophage, we should
ask what differentiates the macrophages of the transient
dust alveolitis from that of the normal lung or that of a
fibrosing alveolitis? Firstly, the macrophage of the normal lung is a permanent component of the lung defence
mechanisms which, under normal healthy conditions, is
in a quiescent state, producing minimal amounts of
secretions [57, 58].
The deposition of the particles in the lower respiratory
tract initiates in situ chemotactic activity for the
macrophage which accumulates at the site of particle
deposition [51]. Basal metabolic activity is increased, it
displays pronounced ruffling of surfaces, phagocytosis
and cell size increases, function is enhanced and, usually
within hours, most particles deposited are phagocytosed
[48-50]. If the particle or fibre phagocytosed is "inert",
limited secretory activity of the alveolar macrophages
87
occurs, the macrophage alveolitis is transient with little
or no lung tissue scarring [55-57). Under the circumstances of exposure to inert dusts, alveolar macrophages
rapidly phagocytose the vast majority of particles but
they do not increase secretion of molecules known to
contribute to the pathogenesis of chronic lung disease
[54-56).
Exposure of the lower respiratory tract to fibres or
particles known to cause lung injury, bioactive, can lead
to the following lung tissue responses:
1. The no-retention reaction: most fibres appear to be
cleared away from the lung tissue rapidly without significant lung tissue scarring. This is the case in several
experimental conditions of low exposures to chrysotile
[59). It is also the case in some long-term asbestos workers
which may be estimated in our clinical work to be up to
50% of all workers [4-6]. This has been observed in the
animal model [59] and in long-term chrysotile miners
[10].
2. The low-retention reaction: fibre retention is low because of the so-called "individual susceptibility factor",
lung tissue reaction is limited to the site of deposition
where macrophages accumulate and eventually are replaced by fibrotic scars limited to the distal airways [10,
23). In this study it appears to be the case in our sheep
in group A.
3. The high-retention reaction: fibre retention is significantly higher, lung tissue reaction is more intense, a
diffuse macrophagic alveolitis can be recognized. The
secretory activity of macrophages is enhanced. Oxidant
production is increased [42], fibronectin production
[22, 41, 42, 54], neutrophil chemotactic activity [60, 61]
and fibroblast growth activity [42, 62] are increased, plasminogen activator [63] and interleukin 1 [64] are secreted at higher levels. If these secretions are sustained,
diffuse lung damage occurs with the development of
chronic interstitial lung disease. The latter observation
has been particularly well documented in animal models.
This high-retention reaction is probably the case of
individuals with interstitial lung disease associated with
chronic inhalation of inorganic dust, asbestos in particular [41, 42]. The men with asbestosis have a high fibre
content of their lung tissue [10-15] and a well-described
fibrosing alveolitis where macrophages are clearly activated [5, 41, 42, 61). This type of reaction has also been
well characterized in animal models. In the sheep model
[22, 27], this high retention reaction has all of the secretory characteristics reported in humans and becomes a
progressive interstitial lung fibrosing disease even in the
absence of further dust exposure. The animal models have
demonstrated that these macrophage secretions needed
to be sustained to initiate and maintain the development
of a fibrosing lung process.
In this paper, we report on sheep which developed a
relatively less intense alveolitis than in our previous
experiments but, nonetheless, a diffuse reaction of the
lung to chrysotile exposure. The sheep in group B had
diffuse lung infiltrates; their BAL analyses documented
an expanded cell population of the lower respiratory tract
(fig. 2). Furthermore, there was evidence of enhanced
secretory function of the cells: oxidant production was
88
R. BEGIN, A. CANTIN, P. SEBASTlEN
increased, fibronectin production and accumulation was
increased; LDH release was increased and albumin was
accumulating slightly in the alveolar space. This alveoIitis has been shown previously in the sheep model to
enhance Gallium-67 lung uptake [4]. However, this
reaction of modest intensity did not significantly alter
fibroblast growth activity or increase procollagen 3 of
lung lavage as previously documented in the presence of
fibrosing lung disease [41, 42] and, thus, could at least
"in theory", regress, leaving minimal lung damage.
This observation is of interest as we have previously
documented enhanced prostaglandin E2 activity in the
sheep model at the stage of early asbestosis [65), a
prostaglandin which is capable of inhibiting fibroblast
growth [66). The prostaglandin E2 secretion by fibroblasts could be induced by alveolar macrophages and
inhibit the fibrotic lung response [67).
This observation of an intermediate lung reaction
between the "low- and the high-retention reactions"
occurring after exposures to dusts with known fibrogenic
potentials is important as it describes a potentially
reversible/non-scarring reaction if exposure is stopped.
In addition, the present study documents that the chrysotile
induced alveolitis at the time of initial radiographic
detection has an incompletely expressed fibrosing activity. It could provide an explanation for the clinical observation of non-progression/regression of several cases of
minimal asbestosis in humans.
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Les exposition a l'asbeste chrysotile peuvent provoquer une
alveolite avec activite fibrosante Iimitee dans un sous-groupe
de moutons avec tendance elevee a retention des fibres. R.
Besin, A. Cantin, P. Sebastien.
RESUME: Des observation anterieures de notre laboratoire et
d'autres institutions ont mis en evidence que !'inhalation de
poussiere minerale fibrogene peut stimuler la production de
fibronectine par les macrophages alveolaires (Fn) et augmenter
l'activite de croissance des fibroblastes dans le liquide de lavage pulmonaire (FGA). Pour investiguer les relations entre ces
modifications et la retention de fibres, et le developpement
d'asbestose, nous avons expose 15 moutons a 100 mg de fibres
de chrysotile canadien dans 100 ml de solution saline a 10
jours d'intervalle, et 10 moutons a 100 ml de solution saline
aux memes intervalles. Les anirnaux ont ete etudies a des intervalles de 3 mois par cliche thoracique, examen fonctionnel
pulmonaire (PFf) et lavage pulmonaire (BAL ). Au 18e mois,
10 moutons avaient des anomalies au clincM thoracque (categoric ~ 1) (groupe B), et 5 moutons avaient des radiographies
normales (categoric 0) (groupe A). Les epreuves fonctionnelles
ont mis en evidence des types restrictifs d'anomalies dans les
deux groupes, plus severcs toutefois dans le groupe B. Les
analyses du B AL ont mis en evidence que les moutons du
groupe A avaient une cellularite et un examen biochimique
comparables a ceux des controles; par contre, les moutons du
groupe B avaient des augmentations significatives du nombre
total de cellules dans le BAL (x 2) des macrophages (X 2), des
neutrophiles (x 4), et des eosinophiles (x 3). On notait en outre
une augmentation de la lactate dehydrogenase du BAL, une
augmentation de Fn, alors que FGA et le procollagene 3 etaient
comparables aux controles. La retention des fibres est significativement augmentee chez tous les animaux exposes, et 2.5
fois plus elevee dans le groupe B que dans le groupe A m algre
des expositions sirnilaires (70 infusions intra-trachCales de 100
mg de chrysotile). La stimulation de la rentetion des fibres dans
le groupe B a precede !'apparition de la maladie. En conclusion, cette etude confrrme nos observations anterieures, qui
reliaient la susceptibilite individuelle a developper une alveollite a la charge individuelle en poussiere, et qui documentaient
que l'exces de retention de fibres peut etre observe avant que
la maladie ne soit detectable. En outre, nous avons mis en
evidence une alveolite precoce induite par le chrysotile avec
une activitc fibrosante incompletement exprirnee au moment de
la detection radiographique initiale. L'intensite de l'alveoltie est
en relation avec le degre de retention de fibres.
Eur Respir J., 1990, 3, 81-90.