Special neutrophil elastase inhibitory activity ... patients with silicosis and asbestosis

Eur Respir J
1989, 2, 751-757
Special neutrophil elastase inhibitory activity in BAL fluid from
patients with silicosis and asbestosis
A. Scharfman, A. Hayem, M. Davril, D. Marko, M.H. Hannothiaux, J.J. Lafitte
Special neutrophil elastase inhibitory activity in BAL fluid from patients with
silicosis and asbestosis A. Scharfman, A . Hayem, M. Davril, D. Mar/w, M.H.
Hannothiaux, J. J. Lafitte.
ABSTRACf: Pneumoconiosis Is defined as the disease resulting from a
chronic exposure to different Inorganic dusts. ln order to assess the lung
defence against the effects of dust exposure, we studied the bronchoalveolar lavage (BAL) fluids from 30 silicotic patients (9 of them having a
diagnosis of progressive massive fibrosis (PMF)) and 8 subject<; with a
diagnosis of asbestosis. Total protein content, N-acetyl-P-0-glucosamlnidase
activity, free elastase-Like activity, Immunoreactive a 1-prot.elnase l.nJtlbltor
(a .fl) and neutrophil elas tase Inhibitory capacity (NE!C) were determined,
and the values obtained were compared to those or 14 control DAL fluids.
In all of the patients, our data showed a significant Increase of total protein
content and free elastase-like activity. In contrast, N-acetyl-P-Dglucosamlnldase activities did not reacb statistical slgnlncance. Values concerning Immunoreactive a 1PI and NEIC were significantly raised only In
patients with PMF and with asbestosis. When the ratio NEIC/
Immunoreactive a 1PI was calculated, a significant difference wa.c; noticed
In the asbestosis group; on the other hand, thLo; ratio was sfgnirlcantly
reduced lo the group of PMF patients. After neutrophil ela.o;tase addition,
an electrophoretic study by SDS-PAGE and lmmunoblottlng was carried
out; lt sbowed more proteolysed a 1PI In tJ1e BAL fluids having a lowered
NEIC/a1PJ ratio. These facts could be e~-plalned by the presence or InhibItors of neutrophil elastase different from a 1 1'J.
Eur Respir J., 1989, 2, 751- 757
Pneumoconiosis is defined as a disease resulting from
chronic exposure to inorganic dust. The common inorganic compounds involved are silica alone or mixed with
coal and airborne asbestos. The main consequence of
chronic silica inhalation is a fibrotic reaction in the lung,
however, the mechanism leading to lung fibrosis is
unknown and it is puzzling that only some exposed
subjects develop a progressive massive fibrosis (PMF)
[1]. Prolonged inhalation of airborne asbestos may lead
to the deve lopmc m of a form of inters titial lung fibrosis
(called asbestosis) or o ther non-fibrotic diseases.
RoM er al. [2] demonstrated that chro nic inhalation of
inorganic dusts induces an inflammation of the lower
respiratory LCact. The innammatory process is do minated
by alveolar macrophages that arc releasing excessive
amounts of media tors. Despite the fact that asbestos, coal
and s ilica are very different agents, alveolar macrophages release similar mediators. In contrast, DoNALDSON et
al. [3) demonstrated that in the rat the pattern and magnitude of the response to inhalation of quartz and
c hrysotile as bestos arc different.
We, therefore, investigated whether the bronchoalveolar lavage (BAL) flui dc; of patients suffering from
silicosis with and without PMF or asbestosis were differ-
Unite lNSERM No. 16, Place de Verdun, 59045 Lille
Cedex, Prance.
Correspondence: A. Hayem, Unite lNSERM No 16,
Place de Verdun, 59045 Lille Cedex, France.
Keywords: Bronchoalvcolar lavage; defence; elastase;
inhibitory capacity; pneumoconiosis.
Received November 1, 1988; accepted for publication April 13, 1989.
Supported by La Communaute Europeenne CharbonAcier (grant number 7248.33.017)
ent. We chose to focus our study on the biochemical
constituents which were previously shown to be
important in BAL fluids such as the activity of
N-acetyl-~-D-glucosaminidase which reflects cell activation, free elastase-like activity which seems to be
related to the evolution of the disease [4-8), and o.1proteinase inhibitor (all) and neutrophil elastase inhibitory capacity (NEIC) which reflect lung defence against
proteolytic injury.
Material and methods
Patient and control populations
The patient population was composed of two groups
of patients suffering from occupational lung diseases.
Since cigarette smoki ng causes inflammation of the lower
respiratory tract, inhibits asbestos clearance [9] and
therefore worsens asbestosis [10), we evaluated on ly
nonsmoking subjects.
The ftrst group was composed o f 30 patients with
silicosis (27 men who were coal workers and three women
working with abrasive powders). The subjects with
752
A. SCHARFMAN ET AL.
silicosis were divided into two sub-groups: 21 patients
with simple silicosis and the 9 patients with a diagnosis
of massive fibrosis (PMF). The diagnosis was based on
radiological findings according to the Bureau International du Travail.
The second group consisted of eight subjects having a
diagnosis of asbestosis (all male). The diagnosis was based
on the history of exposure, on radiological findings and
the characterization of the fibres.
The conttol population was composed of 14 nonsmoking subjects, four of them were healthy volunteers, the
others underwent routine fibreoptic bronchoscopy. All
the controls had normal pulmonary function tests and
chest X-rays.
Informed consent was obtained from all subjects.
Bronchoalveolar lavage
Bronchoalveolar lavage (BAL) was performed in
Calmeue Hospital, Lille, using 250 ml of sterile saline
solution in aliquots 5x50 ml [11], with immediate gentle
vacuum aspirations after each aliquot. The fluid recovered from the first aspiration was discarded because it is
thought to be representative of the bronchial level [12,
13). The fluids from the other aspirations were pooled
and immediately centrifuged at 800 g for 10 min. The
supematant fluid was frozen in aliquots for later assays.
Cell analysis
The cell pellet obtained after centrifugation of the
bronchoalveolar lavage was washed twice with Hank's
medium. The total cells were counted in a haemocytometer chamber and the differential count on smears
stained by the May-Griinwald method. The results of
the cell count were expressed as the total number of cells
for the recovered fluid. The relative amount of each type
of cell was expressed as a percentage of cellularity.
Biochemical analysis
The determinations were all performed on native
unconcentrated fluid in duplicate. The results were
expressed per ml of recovered BAL fluid.
Protein content was measured by the Coomassie Blue
method [14] using human serum albumin as the standard. The results were expressed as J..lg·mJ·'.
N-acetyl-P-D-glucosaminidase activity was determined
using 4-methylumbelliferyl-N-acetyl-P-D-glucosaminide
as described previously [4). Enzymatic activity was
expressed as nmol of substrate released·min·'·ml: 1 (arbitrary units).
Free elastase-like activity (amidolytic activity) was
quantitated using the synthetic substrate succinyl(trialanyl)-paranitroanilide (SLAPN, Biosys) after a 2 h
incubation at 37°C with the BAL fluids [6). In the same
way, the activity of dilutions of a solution of neutrophil
elastase was evaluated and compared to those of BAL
fluids. The neutrophil elastase was purified from purulent sputum according to MARTODAM et al. [15] and found
to be more than 90% active by active site titration using
published kinetic constants [16]. Therefore, the free
elastase-like activity was expressed as neutrophil elastase equivalent (l0·'3mol·mP).
Immunoreactive a,-pror.ease inhibitor (a1PI) was
determined by immunoncphelomctry-laser as previously
described [17]. A Hyland-laser nephelometer PDQ
(Hyland Laboratories) was used with procedures recommended by the manufacturers (Hyland a,PI-antibody and
Hyland-tcst standard A). This method was shown to give
accurate results even if BAL a 1PI is in a complexed
and/or a proteolysed form [17).
The use of some commercial standards for a , PI evaluation was recognized as yielding overestimated values
by radial immunodiffusion [18). Since an international
standard is not yet available, we standardized the Hyland
all standard with serum all prepared in the laboratory
[19], the purity of which was checked by immunoelectrophoresis and analysis in SDS-PAGE. The preparation
was quantified by the Lowry method. Dilutions of the
a 1PI solution were prepared with Lhe working buffer
(10 mM sodium phosphate pH 7.4, containing 0.14 M
NaCl, 2 g% Tween 20 and 40 g% polyethyleneglycol Mr
6,000) with 0.050 g% human serum albumin in order to
take into account the protein composition of the BAL
nuids. The concentration of a 1PI solution was calculated
from the nephelomelric measurements of a 1PI dilutions:
it was 10% lower Lhan that determined by the Lowry
method. Taking into account the variation coefficients of
the nephelometric method [17) (within-day reproducibility and day-to-day precision), we considered the BAL
measured a,PI values as the true values; they were
converted in mol·ml-1 using a Mr equal to 52,000.
The neutrophil elastase inhibitory capacity (NEIC) was
measured by hydrolysis of L-pyroglutamyl-L-prolyl-Lvaline-p-nitroanilide (S 2484, Kabi diagnostica) as previously described [20). Briefly a constant amount of
neutrophil elastase (0.05 J.!M) was incubated for 10 min
at 25°C with increasing amounts of BAL fluid in saline
buffer (sodium phosphate 10 mM pH 7.4, NaCl 0.3 M).
After addition of the substrate (final concentration 0.45
mM) the residual activity was measured at 410 nm. The
NEIC was calculated from the point of functional equi valence (determined by linear regression) and expressed as
moles of elastase inhibited·ml·' BAL fluid.
The electrophoretic studies were carried out according
to LAEMMLI [21) i11 a 5- 25% polyacrylamide gradient
slab gel in the presence of SDS (SOS-PAGE). The
dimensions of the gels were ISO x 150 x lmm. Before
being electrophoresed, the BAL fluids were analysed for
protein and a,PI contents. Volume samples were calculated in order to separate 20 ~g protein and loaded by
refill if necessary. Neutrophil elastase was added to the
BAL fluids, in order to have about a 1.5 fold excess over
a 1PI content (mol·mi·'). The mixtures were incubated for
15 min at room temperature before the beginning of the
electrophoresis procedure. Proteins separated by SOSPAGE were transferred electrically to nitrocellulose paper
using an LKB Multiphor li Novablot, with the
BAL IN OCCUPATIONAL LUNG DISEASE
753
discontinuous buffer system recommended by the manufacturers (anode solution pH 10.4: Tris 0.3 M, 20%
methanol, cathode solution pH 7.6: 6-amino-n-hexanoic
acid 40 mM, 20% methanol).
The nitrocellulose sheets were stained by an immunoperoxidase method [22] using 4-chloro-1-naphthol as the
developer [23]. The antibodies against a 1PI were from
Dako. The antiserum against neutrophil elastase was
obtained by immunizing rabbits with neutrophil elastase
purified from purulent sputum according to MARTODAM
et al. [15].
N-acetyl-P-D-glucosaminidase
Statistics
Free elastase-like activity was detected in 3 out of the
14 controls, in 14 out of 21 patients with simple silicosis,
in 4 out of 9 patients with PMF, and in 6 of the 8 patients
with asbestosis. Under our experimental conditions (2 h
incubation), all the values obtained were very low (10·13
mol·ml'1) in controls as well as in patient fluids. However, the differences between patients and controls were
significant (table 2).
The results were expressed as mean±standard deviation. Significance of differences between groups was
determined by Student's t-test When correlations between
two variables were found, a linear regression was calculated. Significance was determined at p<0.05.
For N-acetyl-P-D-glucosaminidase, the values obtained
for all patients were higher than those of control subjects
(table 2). However, the differences were not significant.
It should be noted that the values corresponding to PMF
patients did not significantly differ from those of the
other silicotic patients.
Free elastase-like activity
Results
General characteristics of BAL
Invnunoreactive afPI
The amount of fluid obtained in subjects with silicosis
was decreased as compared to the controls, but was not
significantly different in asbestosis patients (table 1).
In order to establish the mean values for immunoreactive a 1PI we did not use results <1.5xl0'11 mol·ml·1 since
under our experimental conditions, this value represents
Table 1. - General characteristics of bronchoalveolar lavage
Recovered
Number of
fluid. (ml)
cells·m1' xlO"
Macrophages
Controls
157±27
n=14
6.5±2.4
n=lO
87±7
n=6
12±6
1±1
Siljcosis
118±37
n=30
p<0.001
24±21
n=23
p<0.005
85±12
n=20
9±7
6±11
NS
NS
NS
111±52
n=8
15±6
n=8
81±16
n=6
15±12
4±4
NS
NS
NS
NS
NS
Asbestosis
1
Differential cell count %
Lymphocytes
Neutrophils
Ns: non significant. Statistical evaluations were calculated from patients versus controls.
The number of total cells was significantly higher in
the patients with silicosis when compared to controls
(table 1), while no differences were found in the
asbestosis patients. The percentages of macrophages and
Iymphocytes in each disorder were similar to that of
controls. Even if the mean percentage of neutrophils was
elevated in both groups of patients, the differences
between patients and controls did not reach statistical
significance.
Protein content
Total protein content was significantly elevated for all
of the patients (table 2). No differences were noted
between the different groups of patients.
the lower detection limit of the method. A concentration
<1.5x 10' 11 mol·ml' 1 was observed in the BAL of six
control subjects and one patient with simple silicosis. In
patients with silicosis with and without PMF, the mean
value of immunoreactive all was higher than in controls (table 2). There was no significant difference for
patients with simple silicosis, while for the group of PMF
patients the difference was significant. A significant
difference was also observed in the patients with asbestosis. A positive and significant correlation was found
between total protein content and immunoreactive a 1PI
in the patients with simple silicosis (p<0.02) and in the
patients with PMF (p<0.02). On the contrary no correlation was found for these two parameters in the patients
with asbestosis.
754
A. SCHARFMAN ET AL.
Table 2. - Biochemical analyses of bronchoalveolar lavage fluids
Controls
n:=l4
Silicosis
Simple
n=21
Total protein
N-acetyl-f.\-Dglucosaminidase
Free elastase-like
activity
Immunoreactive
ex ll
Neutrophil elastase
inhibitory capacity
)lg·mt 1
units
1Q·13 mol·mJ·I
10· 11 mol·mJ· 1
10-11 mol·m1· 1
48±24
81±52
p<0.02
1±0.7
4±6
2±2
22±25
NS
p<0.02
1.7±0.5
<1.5
n=8
1.9±0.3
n=6
n=l2
<1.5
n:o:l
3.3±2.9
n=20
2.6±2
NS
NS
PMF
n=9
118±57
p<O.OOl
1.8±1.2
Asbestosis
n =8
132±95
p<0.005
1.8±1.7
NS
NS
15±18
p<0.05
5±3.7
p<O.Ol
29±31
p<O.Ol
3.3±1.4
p<0.05
3.3±2.3
p<0.05
4±1.3
p<O.OOOS
«ll: a.1-proteinase inhibitor; PMF: progressive massive fibrosis. All the statistical evaluations
were calculated from patients versus controls.
NS: non significant;
Neutrophil elastase inhibitory capacity
The values obtained in the group of patients with simple
silicosis were not significantly different from controls,
while PMF patients as a group showed significantly
elevated values when compared to controls (p<0.05).
NEIC values in patients with asbestosis were also significantly higher than controls (p<0.0005). In order to
assess the part taken by cxll in the NEIC, we calculated
the ratio NEIC/cx1PI (mol neutrophil elastase inhibited·ml·
1
/mol immunoreactive a 1PI·mi·1) in controls and patients
having an cxll> 1.5xl0'11 mol·ml·1 (six controls, twenty
simple silicosis, nine PMF patients and eight asbestosis).
The results are graphically expressed in figure 1. On
average, no difference was seen between controls and
patients with simple silicosis as a group. This fact must
be due to a very high value (2.90) obtained in one patient.
In contrast, the PMF values were lower than control
values (p<0.05), while in the asbestosis group, they were
significantly higher than controls (p<0.05). These values
were also significantly high when compared to the simple
silicosis group (p<0.02) and the PMF group (p<O.OOl).
Electrophoretic study
added neutrophil elastase: the presence on the
immunoblots of a newly formed complex between all
and elastase would give information about the cx1PI
inhibitory capacity.
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The electrophoretic study was first carried out to investigate the state of a 1PI in the BAL 1uids, since a 1PI
could be found either unaltered or pr:11.eolysed or complexed to some proteases [24]; these latter species have
a modified Mr and are therefore easily located after SDSPAGE followed by immunoblotting. Secondly, the same
techniques allowed us to explore the cx1PI ability to inhibit
Controls
Silicosis
Asbestosis
Fig l. - Graphic representation of lhe ratio NEIC/a1PI eJtpressed as
mol. neutrophil elastase inhibited per ml!mol·immunoreactive a.PI
per ml in BAL fluids from controls, patients wilh simple silicosis (e)
PMF patients (0) and patients wilh asbestosis. The horizontal bars
represented lhe mean values (Controls: 0.9±0.31; simple silicosis:
0.85±0.58; PMF: 0.57±0.23; asbestosis: 1.32±0.35).
755
BAL IN OCCUPATIONAL LUNG DISEASE
Figure 2A shows the results obtained when analysing
a BAL fluid in which a 1PI was thought to be largely
inactive (NEIC/a1PI ratio equal to 0.6). Before addition
of neutrophil elastase, a major component (b) was seen,
identified to native a 1PI by its Mr; after addition of
neutrophil elastase in excess, formation of a complex
between all and elastase was demonstrated by the
presence of a new band (a) revealed not only by antia1PI antibodies but also by anti-elastase antibodies. Many
degraded products revealed by anti-all antibodies were
seen, one of them being the proteolysed form of all
(c); all had not been prevented from degradation by
the presence of other active inhibitors. In contrast, BAL
fluid analysed in figure 2B, obtained from an asbestosis
patient, had an NEIC/a1PI ratio equal to 1.18. After
addition of neutrophil elastase in excess, the complex
all-elastase was easily seen; the only a 1PI degraded
product observed was the proteolysed form (c), which is
normally formed when inhibition of neutrophil elastase
by <xll occurs [25]. This observation brought new evidence of the presence of some other neutrophil elastase
inhibitor(s) different from <x1PI.
A
1
+NE
2
+NE
B
d-+NE
+NE
Fig. 2. - Irnmunoblots of two BAL fluids revealed anti·tt1PI antibodies (1) and anti-neutrophil elastase antibodies (2). In A, BAL fluid
from a silicOtic patient had a NEIC/tt1PI ratio equal to 0.6. In 8, SAL
fluid from an asbestosis patient had a NEIC/tt,PI ratio equal to 1.18.
Samples were electrophoresed from top to bottom. a: elastase· tt.PI
complex; b: native tt.PI; c: proteolysed tt1PI d: elastase in excess;
+NE: with neutrophil elastase added; High molecular weight binding
is also seen when using non-immune rabbit serum (data not shown).
Discussion
The present study demonstrated that in BAL fluids
from patients with asbestosis and silicosis, the same
modifications were obtained for protein content,
N-acetyl-~-D-glucosaminidase, and free elastase-like
activity. In contrast, <x?I and NEIC seemed to vary
specifically with the different diagnoses.
The protein content of BAL fluids of patients is increased, this may just be an indicator of the altered pulmonary epithelium [26]. Local synthesis may a lso be
involved [27, 28].
Glycosidases possess biological activities against many
of the structural components of pulmonary tissue [29];
therefore, they may be of importance in the pathogenesis
of lung diseases, such as pneumoconiosis. Our data show
a non-significant increase of N-acetyl-~-D­
glucosaminidase activity in the BAL fluids of all the silicotic patients. However, we showed in a previous work
that glycosidases were significantly increased in BAL
fluids from nonsmoking pneumoconiotic coal-workers
(p<0.05) (4]. Similar results were also obtained by others
in asbestosis [7]. These discrepancies could be explained
by the low values found in seven BAL fluids from the
silicotic group and two BAL fluids from the PMF group.
The free elastase-like activity we detected in the BAL
fluids of patients was significantly higher than in controls, but the values were very low when compared to
the NEIC of these BAL fluids. Despite major methodological differences in lavage procedures, as well as the
manner of determining the enzyme activity, our results
are in good agreement with other papers [30-32], i.e. the
elastase-like activity is very low in BAL fluids from
healthy nonsmokers, but it is impossible to compare our
values with those of other authors. This elastase-like
activity probably originates from neutrophils. However,
it was significantly increased in all patient BAL fluids,
whereas the neutrophil counts were not significantly
modified. This activity may also derive from macrophages [31) and be representative of the activation state of
these cells. In fact, silica and asbestos fibres induce a
perpetual recruitment of macrophages in the lung ~d
newly arrived macrophages differ from other subpopulations in their biological properties, such as secretion of
enzymes [33]. The separation of macrophage subpopulations followed by the study of their biological activities
will be of interest when studying pneumoconiosis.
Immunoreactive <x1PI measurements gave a wide range
of values and significant differences were obtained only
for the group of PMF patients and for the patients with
asbestosis. In fact, the immunoreactive all was on
average lower in asbestosis than in the silicotic patients
having PMF. a 1PI concentration is dependent on the
plasma concentration and the degree of pulmonary
inflammation leading to an increased transudation [34].
In all the patients with silicosis, immunoreactive a 1PI
content was related to total protein content, in favour of
an increased transudation. In contrast, in asbestosis,
protein and immunoreactive a 1PI contents were not
related; this fact may be the consequence of local synthesis of other proteins such as immunoglobulins [28].
A. SCHARFMAN ET AL
756
The values obtained for NEIC in silicotic patients with
PMF, and patients with asbestosis were significantly
higher than in controls. NEIC is representative of all the
inhibitors present in BAL fluid. a 1PI was reported to be
the major anti-neutrophil-elastase found at the alveolar
level in normal subjects [35]. However, recently several
supposedly distinct inhibitors have been described in
healthy subjects [36] as well as in bronchitic [37] and
a.PI deficient patients [38].
Even in nonsmoker control BAL fluids, all was shown
to be partly inactive against neutrophil elastase [39, 40].
Therefore, the values of the molar ratio NEIC/o. PI are
normally lower than 1. In fact, BoUDIER et al. d6J recently demonstrated a heterogeneity in the composition
of lung anti-elastases, by evaluation of the ratios NEIC/
a1PI: in some subjects, NEIC/o.li ratios were higher
than unity, in others they were equal or lower than unity.
Our data showed the same kind of results. For the controls, the value of the ratio was in good agreement with
that described by BouoiER et al. [36] and very close to
that given by AFFORD et al. [41], although our technical
conditions differed from theirs; according to MoRRISON et
al. [42] they should lead to an underestimation of the
NEIC values that we obtained. This ratio was significantly lower in PMF subjects in contrast to those with
simple silicosis. On the other hand, the mean value of
this ratio in the patients with asbestosis was higher than
in controls. The results in figure l showed that the eight
patients with asbestosis had a ratio higher than the mean
value in controls, while in PMF patients, all but one had
a value lower than the mean control value.
Therefore, it seemed that patients with asbestosis had
a high protection against neutrophil elastase at the alveolar level, not totally due to o.1PI: the electrophoretic study
that we carried out demonstrated a functional activity of
only a part of immunoreactive o..PI. Therefore, there is
no doubt that other inhibitors of neutrophil elastase are
present at the alveolar level, one of them being the human
mucus proteinase inhibitor (or "bronchial inhibitor") but
it represents only 14% of 0.1PI molar concentration [36].
Up to now, the other inhibitors are not well-defined; their
presence at different concentrations would explain why
the defence against neutrophil elastase injury is high only
in some controls, some patients with simple silicosis and
patients with asbestosis. In asbestosis when emphysemataus lesions are seen, they are from tractional origin; the
elastic framework remains intact [43], and this may occur
in part because in this disease anti-neutrophil elastase
defence is high.
Acknowledgements: The authors greatly thank J>rof Voisin
and his team for providing them with the BAL fluids and the
information concerning the patients. The writers also thank M.P.
Ducourouble and E. Van Brussel for their excellent technical
assistance.
References
1.
2.
RG.
tion
Seaton A.- Coal and the lung. Thorax, 1983, 38, 241-243.
Rom WN, Bitterman PB, Rennard SI, Cantin A, Crystal
-Characterization of the lower respiratory tract inflammaof nonsmoking individuals with interstitial lung disease
associated with chronic inhalation of inorganic dusts. Am Rev
Respir Dis, 1987, 136, 1429-1439.
3. Donaldson K, Bolton RE, Jones A, Brown GM, Robertson
MD, Slight J, Cowie H, Davis JMG. - Kinetics of the bronchoalveolar leucocyte response in rats during exposure to equal
airborne mass concentrations of quartz, chrysotyle asbestos, or
titanium dioxide. Thorax, 1988, 43, 525-533.
4. Sablonniere B, Scharfman A, Lafitte JJ, Laine A, Aerts C,
Hayem A. - Enzymatic activities of bronchoalveolar lavages in
coal workers pneumoconiosis. Lung, 1983, 161, 219-222.
5. Hayem A. Scharfman A, Laine A, Lafitte JJ, Degand P. Le lavage bronchoalveolaire dans la pneumoconiose du mineur
de charbon. Aspects biochimiques. Rev Fr Mal Respir, 1983,
11, 417-426.
6. Scharfman A, Hayem A, Marko D, Lafitte JJ, Fressier B,
Mizon J. - Study of some biochemical parameters involved in
the inactivation of a 1-proteinase inhibitor. /n: Pulmonary
emphysema and proteolysis. J.C. Taylor, C. Mittman eds,
Academic Press, New York 1987, pp. 473-480.
7. Jaurand JC, Gaudichet A, Atassi K, Sebastien P, Bignon
J. -Relationship between the number of asbestos fibres and the
cellular and enzymatic content of bronchoalveolar fluid in
asbestos exposed subjects. Bull Eur PhysiopaJhol Respir, 1980,
16, 595~06.
8. Dethloff LA, Gladen BC, Gilmore LB, Hook GER. Quantitation of cellular and exttacel!ular constituents of the
pulmonary lining in rats by using bronchoalveolar lavage. Am
Rev Respir Dis, 1987, 136, 899-907.
9. McFadden D, Wright JL, Wiggs B, Churg A. - Smoking
inhibits asbestosis clearance. Am Rev Respir Dis, 1986, 133,
372-374.
10. Tron V, Wright JL, Harrison N, Wiggs B, Churg A. Cigarette smoke makes airway and early parenchyma!
asbestos-induced lung disease worse in guinea-pig. Am Rev
Respir Dis, 1987, 136, 271-275.
11. W allaert B, Hatton PY, Grosbois JM, Tonne! AB, Devulder
B, Voisin C.- Subclinical pulmonary involvement in collagenvascular diseases assessed by bronchoalveolar lavage. Am Rev
Respir Dis, 1986, 133, 574-580.
12. Tonnel AB, Voisin C, Lafitte JJ, Ramon P, Aerts C. Variations des populations cellulaires recueillies par lavage
bronchoalveolaire en fonction de la topographic des lesions et
de l'etage explore. In : Le lavage bronchoalveolaire chez
l'homme G. Biserte, J. Chretien, C. Voisin eds, INSERM, Paris,
1979, pp. 271-280.
13. Sybille Y, Martinot JB, Staquet P, Delaunois 1., Chatelain
B. Delacroix DL.- Antiproteascs are increased in bronchoalveolar lavage in interstitial lung disease. Eur Respir J, 1988, 1,
498-504.
14. Spector T. - Refinement of Coomassie blue method of
protein quantitation. A simple and linear spectrophotometric
assay for 0.5 to 50 )lg of protein. Anal Biochem, 1978, 86,
142-146.
15. Martodam RR, Baugh RJ, Twumasi D, Liener IE. - A
rapid procedure for the large scale purification of elastase and
cathepsin G from human sputum. Prep Biochem, 1919, 9, 15-31.
16. Nakajima K, Powers JC, Ashe BM, Zimmerman N. Mapping the extended substrate binding site of cathepsin G and
human leukocyte elastase. J Bioi Chem, 1979, 254, 4027-4032.
17. El Yamani J, Hayem A, Lafitte JJ, Gressier B, Mizon J.
- Functional and immunoreactive a 1 proteinase inhibitor in
bronchoalveolar lavages: methodological studies. Bull Eur
Physiopathol Respir, 1986, 22, 359-363.
18. Wewers MD, Ca~olaro A, Sellers SE, Swayze SC, McPhaul
KM, Wittes IT, Crystal RG. - Replacement therapy for
alpha 1 -antitrypsin deficiency associated with emphysema. N Engl
J Med, 1987, 316, 1055-1062.
BAL IN OCCUPATIONAL LUNG DISEASE
19. Vercaigne-Marko D, Carrere J, Ducourouble MP, Davril
M, Laine A, Amouric M, Figarel!a C, Hayem A. - "In vivo"
and "in vitro" inhibition of human pancreatic chymotrypsin A
by serum inhibitors. Bioi Chem Hoppe-Seyler, 1987, 368, 37-45.
20. Marko D. Hannothiaux MH, Scharfman A, Hayem A,
Gressier B, Lafitte JJ, Mizon J. - Activite fonctiormelle de l'a1antiprotease des liquides de lavages bronchoalveolaires de sujets
atteints de pneumoconiose.ln: Silicosis and mixed-dusts pneumoconiosis. L. Le Bouffant ed, INSERM, Colloque, 1987, pp.
123-128.
21. Laemmli UK.- Cleavage of structural proteins during the
assembly of the head of bacteriophage T". Nature, 1970, 227,
680-685.
22. Perini JM, Dehon B, Marianne T, Klein A, Roussel P. Reducing the artifacts produced by impure antisera in
immunoblots of low-molecular mass proteins in urine. Clin
Chem, 1986, 32, 811-815.
23. Hawkes R, Niday E, Gordon J. - A dot-immunobinding
assay for monoclonal and other antibodies. Anal Biochem, 1982,
116. 142-147.
24. Stockley RA, Afford SC. - Qualitative studies of lung
lavage cx.J-proteinase inhibitor. Hoppe-Seyler's Z Physiol Chem,
1984, 3o5, 503- 510.
25. Padrines M, Schneider-Pozzer M, Bieth JG. - Neutrophiloxidized cx.1-proteinase inhibitor is a slow-binding inhibitor of
neutrophil elastase. Am Rev Respir Dis, 1988, 137, llOA.
26. Low RB, Davis CTS, Giancola GN. - Biochemical analysis of bronchoalveolar lavage fluids of normal healthy volunteers smokers and nonsmokers. Am Rev Respir Dis, 1978, 118,
863- 875.
27. Calhoun WJ, Christman JW, Bershler WB, Graham WGR,
Davis G. - Raised immunoglobulin concentration in bronchoalveolar lavage fluid of healthy granite workers. Thorax,
1986, 41, 266-273.
28. Gellert AR, Perry D, Langford JA, Riches PG, Rudd RM.
- Asbestosis. Bronchoalveolar lavage fluid proteins and their
relationship to pulmonary epithelial permeability. Chest, 1985,
88, 730-735.
29. Brieland GK, Kunkel RG, Fantone JC. - Pulmonary alveolar macrophage function during acute inflammatory injury.
Am Rev Respir Dis, 1987, 135, 1300-1306.
30. Janoff A, Raju L, Dearing R.- Levels of elastase activity
in bronchoalveolar lavage fluids of healthy smokers and nonsmokers. Am Rev Respir Dis, 1983, 127, 540-544.
31. Niederman MS, Fritts LL, Merril WW, Fick RB, Matthau
RA, Reynolds HY, Gee ffi L. - Demonstration of free elastolytic metalloenzyme in human lung lavage fluid and its relationship to alpha 1-anti-protease. Am Rev Respir Dis, 1984, 129,
943- 947.
32. Fera T, Abboud RT, Richter A, Johal SS. -The effect of
smoking on elastase-like esterase activity and immunologic
neutrophil elastase levels in bronchoalveolar lavage fluid. Am
Rev Respir Dis, 1986, 133, 568-573.
33. Bowden DH.- Macrophages, dust and pulmonary diseases.
Exp Lung Res, 1987, 12, 89- 107.
34. Stockley RA. - Measurement of soluble proteins in lung
secretions. Thorax, 1984, 39, 241-247.
35. Gadek JE, Fell GA, Zimmerman RL, Rennard SI, Crystal
RG. - Anti-elastases of the human alveolar structure. Implication for the protease-antiprotease theory of emphysema. 1 Clin
Invest, 1981, 68, 889- 898.
36. Boudier C, Pelletier A, Oast A, Tournier JM, Pauli G,
Bieth JG. - The elastase inhibitory capacity and the cx.1-
757
proteinase inhibitor and bronchial inhibitor content of bronchoalveolar lavage fluids from healthy subjects. Bioi Chem
Hoppe.Seyler, 1987, 368, 981-990.
37. Stockley RA, Morrison HM, Smith S, Tetley T. - Low
molecular mass bronchial proteinase inhibitor and a 1-proteinase
inhibitor in sputum and bronchoalveolar lavage. HoppeSeyler's Z Physiol Chem, 1984, 365, 587-595.
38. Morrison HM, Kramps J, Bumett D, Stockley RA.- Lung
lavage fluid from patients with cx.1-proteinase inhibitor deficiency or chronic obstructive bronchitis: anti-elastase function
and cell profile. Clin Sci, 1987, 72, 373-381.
39. Boudier C, Pelletier A, Paul.i G, Bieth JG. -The functional activity of CX.1 -proteinase inhibitor in bronchoalveolar
lavage fluids from healthy human smokers and non-smokers.
Clin Chim Acta, 1983, 132, 309-315.
40. Wewers MD, Casoralo MA, Crystal RG. - Comparison of
cx.1-antitrypsin levels and anti-neutrophil elastase capacity of
blood and lung in a pal.ient with the cx. 1.antitrypsin phenotype
null-null before and during CX.1-antitrypsin augmentation therapy.
Am Rev Respir Dis, 1987, 135, 539-543.
41. Afford SC, Bumett D, Campbcll EJ, Cury ID, Stockley
RA. -The assessment of cx.1-proteinase-inhibitor form and function in lung lavage fluid from healthy subjects. Bioi Chem
Hoppe-Seyler, 1988, 369, 1065- 1074.
42. Morrison HM, Kramps JA, Afford SC, Bumett D, StockIcy RA. - The effect of assay conditions on the measurement
of anti-elastase function in lung secretions Clin Chim Acta,
1987, 162, 165-174.
43. Spencer H. - The pneumoconioses and other occupational
lung diseases In: Pathology of the lung, vol 1. H. Spencer ed.,
Pergamon Press, 1985, pp. 413-510.
Comparaison de la capacite inhibitrice vis-a-vis de I'ilastase
leucocytaire des UBA malades aJteints de silicose et d'asbestose. A. Scharfman, A. Hayem, M. Davril, D. Marko, M.H.
Hannothiaux, 1.1. Lafitte.
RESUME: L'inhalation chronique de poussieres inorganiques
est responsable du declenchement de la maladie pneumoconiotique. Afin d'evaluer le potenticl de dCfense, au niveau
alveolaire, nous avons etud ie les Jiquides de lavages bronchoalveolaires (LLBA) de 30 patients atteints de silicose, (9
d'entre eux presentaient une fibrose massive progressive (FMP)
et de 8 malades avec un diagnostic d'asbestose. Sur ces LLBA,
ont ete determines: teneur en proteines, activite de N-acetyl-~0-glucosaminidase, activite esterasique de type elastase, teneur
en cx.1-antiprotease (all) immunoreactive et capacitc inhibitrice vis-~1-Vis de !'elastase leucocytaire (CIEL). Les valeurs
obtenues ont ete comparees a celles de 14 LLBA provenant de
sujets tcmoins. Pour tous les maladcs. on peut constater une
augmentation statistiquement significative de la leneur en
protcines et de l'activite de type elastase. Par co ntre, les valeurs
obtenues pour l'activiu! de N-acetyl-B-D-glucosaminidase ne
sont pas significatives. En ce qu i conceme l'a1PI et la CIEL.
lcs valeurs ne sonL significativement augmentecs que pour les
malades atteints de FMP et d'asbcstose. Le rapport CIELA:t1PI
immunoreactive est significativement eleve chez les sujets
aueints d'asbestose alors qu'il est abaisse chez les malades
atteints de FMP. Lorsque le rapport CIEL/cx.1 PI est largement
inferieur a 1, une etude electrophoretique a montre la predominance des formes proteolysees de l'o:li obtenues apres addition d'elastase leucocytaire en exces.
Eur Respir 1., 1989, 2, 751- 757.