35
Clinical Science ( 1989)77,3541
Effects of plasma, tumour necrosis factor, endotoxin and
dexamethasone on extracellular proteolysis by neutrophils
from healthy subjects and patients with emphysema
DAVID BURNETT, ANITA CHAMBA, SUSAN L. HILL
AND
ROBERT A. STOCKLEY
Lung Immunobiochemical Research Laboratory, The General Hospital, and Departments of Immunology and Medicine, University of
Birmingham, Birmingham, U.K.
(Received 27 July/l4 November 1988; accepted 18 November 1988)
SUMMARY
INTRODUCTION
1. Neutrophils from patients with chronic obstructive
bronchitis and emphysema or age-matched control
subjects were cultured on a substrate of 12SI-fibronectin.
The neutrophils from patients with lung disease digested
significantly more fibronectin and released more elastase
into the culture supernatant than did cells from control
subjects. Preincubation of neutrophils from emphysematous patients with plasma from control subjects significantly inhibited fibronectin digestion by the patients’
neutrophils by, on average, 10%. Preincubation of control
subjects’ neutrophils with plasma from emphysematous
patients had no effect on fibronectin digestion.
2. Tumour necrosis factor increased fibronectin
digestion in a dose-dependent manner when the cytokine
was added to the adherent cells but not when preincubated with the polymorphonuclear leucocytes in suspension. Bacterial endotoxin in concentrations above 6 pg/ml
significantly increased fibronectin digestion by neutrophils, but leukotriene B,, interferon-y and interleukin-1a
had no significant effects.
3. Dexamethasone inhibited fibronectin digestion by
neutrophils in a dose-dependent manner, from 11% at
mol/l to 68% at
mol/l.
Leucocyte proteinases are thought to be responsible for
the tissue damage associated with chronic obstructive
lung diseases [ 1,2]. In particular, neutrophil elastase (NE)
has been shown to produce emphysematous lesions and
bronchitis in experimental animals [3]. The lung usually
contains inhibitors of NE that protect the tissues from this
enzyme. Nevertheless, excessive tissue destruction by
proteinases is believed to occur in some individuals,
leading to chronic lung disease.
Several studies in vitro have shown that polymorphonuclear leucocytes (PMN)can digest protein substrates on
which these cells are cultured [4-71. This process appears
to be due to proteolysis by NE activity in the pericellular
area between the cell and substrate [4, 81 and continues
even in the presence of inhibitors such as a,-antitrypsin
[4-81. The pericellular area therefore appears to be a
privileged site for extracellular tissue degradation. Thus
as PMN migrate through the interstitium of the lung,
extracellular digestion of connective tissue could occur
even in the presence of proteinase inhibitors. Excessive
tissue damage leading to disease may therefore be
dependent upon the degree of PMN recruitment and
activation.
Large numbers of PMN are found in bronchoalveolar
lavage fluid from cigarette smokers and subjects with
chronic bronchitis and emphysema [9, 101. Furthermore,
PMN accumulation has been shown in the alveolar septa
of smokers [ 111. Recent studies have shown that blood
PMN from subjects with chronic bronchitis and emphysema have an enhanced chemotactic response to the peptide N-formylmethionyl-leucylphenylalanine [ 12, 131. In
addition, the PMN were shown to digest more extracellular protein than those from healthy control subjects [ 131.
These observations in vitro suggest a mechanism which
could lead to excessive tissue damage in vivo, resulting in
emphysema.
The reasons for the enhanced extracellular proteolysis
by cells from subjects with lung disease are not known.
Key words: elastase, emphysema, endotoxin, neutrophil,
proteolysis, tumour necrosis factor.
Abbreviations: FN, fibronectin; IFN, interferon- y; IL- 1,
interleukin-1 a; LTB, leukotriene B,; NE, neutrophil
elastase; PMN, polymorphonuclear leucocytes; TNF,
tumour necrosis factor a.
Correspondence: Dr David Burnett, Lung Immunobiochemical Researeh Laboratory, The General Hospital, Steelhouse
Lane, Birmingham B4 6NH, U.K.
36
D. Burnett et al.
One explanation is that blood PMN are activated or
primed by factors released as a result of lung inflammation. Several factors associated with inflammation have
the potential to activate PMN. In the present study we
have investigated the effects of bacterial endotoxin and
several cytokines on extracellular proteolysis by blood
PMN. Since any released activating factors may be
present in the blood, we have studied the effects of plasma
from patients with emphysema on the cells from healthy
control subjects. We also wished to establish whether
enhanced proteolysis is associated with increased release
of NE by PMN.
In a previous study [14] we showed that the elastase
inhibitory capacity of the lung secretions of patients with
chronic bronchitis was increased after corticosteroid
treatment. This effect was apparently not due to increased
concentrations of a ,-antitrypsin and could have resulted
from reduced release of NE within the airways. In the
present study we have therefore investigated the effects of
dexamethasone on extracellular proteolysis by neutrophils.
Patient studies
The studies were approved by the Central Birmingham
Health Authority Research Ethical Committee and all
subjects gave their informed consent.
Eight patients with established chronic bronchitis and
emphysema were investigated. Their mean age was 67
years (range 58-74 years), six were male and three were
current cigarette smokers.
Diagnosis was based on clinical history, radiology and
lung function tests, the results of which were as follows
(values in parentheses are percentages of predicted values
for age and height): forced expiratory volume in 1 s, mean
1.13, SD 0.65 litres (43.2, SD 19.9%); forced expiratory
volume in 1 s/forced vital capacity, mean 38.5%, SD
13.6% (56.2%, SD 2 1.2%);total lung capacity, mean 6.64,
SD 1.3 litres ( 114%, SD 13.4%); residual lung volume/total
lung capacity, mean 53.4%, SD 10.7% (137%, SD 31%);
transfer factor per unit lung volume, mean 1.1, SD 0.4
mmol min- I k h - litre (79%, SD 26%). All patients were
clinically stable with no evidence of an overt infection.
Eight healthy control subjects were recruited from
hospital staff. Their mean age was 56 years (range 49-58
years). All were male and five were smokers. All had lung
function tests within the normal ranges predicted for age
and height and none had a history of chronic disease or an
overt infection.
’
Isolation of blood neutrophils. All reagents were
assayed for endotoxin using the Kabi Vitrum Coatest
obtained from Flow Laboratories (Rickmansworth,
Herts., U.K.) and contained less than 20 ng/l.
Venous blood (10 ml) was collected from each subject
into lithium heparin anticoagulant. The blood samples
were diluted with an equal volume of 0.15 mol/l NaCI.
Each sample was layered on to 2 ml of 54% (w/v) Percoll
(density 1.075 g/ml; Pharmacia AB, Uppsala, Sweden)
which had itself been layered on to 3 ml of 78% (w/v)
Percoll (density 1.096 g/ml). The tubes were centrifuged
at 200 g for 25 min and the PMN at the interface of the
two Percoll densities were collected, washed three times
with
4-(2-hydroxyethy1)-1-piperazine-ethanesulphonic
acid-buffered RPMI 1640 medium (Flow Laboratories,
Rickmansworth, U.K.) and counted before resuspension
in RPMI 1640 medium.
Extracellular fibronectin (FN) proteolysis. The assay
for 12%FN proteolysis by PMN was based on that
described by Campbell et al. [4]. Purified human plasma
FN was a gift from Mr R. Drew, Department of I b u n ology, University of Birmingham, and was iodinated by
the chloramine-T method with sodium [1251]iodide
obtained from Amersham International, Amersham,
Bucks, U.K. The labelled protein was diluted with a solution of unlabelled FN in 0.05 mol/l sodium carbonate/
bicarbonate buffer, pH 9.6, to give 2000 c.p.m./pg of FN.
The FN solution was dispensed into NUNC microtitre
plate wells (Gibco, Paisley, Scotland, U.K.). Each well was
filled with 0.2 ml of solution containing a total of 30 pg of
FN (60000 c.p.m.). The plates were allowed to dry at
37°C and were washed with phosphate-buffered saline,
pH 7.2, to remove non-adherent FN. The PMN suspensions (3 x 10’ cells on 0.2 ml of RPMI 1640 medium) were
applied to the wells and the plates incubated for 3 h at
37°C in a humidified atmosphere of 5% CO,/air. Control
wells contained no cells. After incubation, the contents of
each microtitre well were harvested, centrifuged and the
supernatant collected. The amount of solubilized FN in
each supernatant was assayed by the measurement of 12sI
using an LKB Multigamma I1 gamma counter. Each cell
preparation was assayed six times and the mean value was
calculated. The results were expressed as pg of FN
solubilized/3 x lo5 cells. Differences in the amounts of
FN solubilized by PMN from the control subjects and
patients with lung disease were tested using Student’s ttest. The effects of preincubation of PMN with autologous
or homologous plasma were tested using Student’s t-test
for paired data.
NE release. The supernatants collected after PMN
( preincubated with autologous plasma) had been cultured
on FN were assayed for NE using the synthetic substrate
succinylalanylalanylalanyl-p-nitroanilide. Each supernatant (25 p l ) was mixed, in a microtitre plate well, with
0.02 pg of substrate dissolved in 0.2 ml of 0.05 mol/l
Tris-HCI buffer, pH 8.6 with 0.1% (v/v) Triton X-100.
The plates were incubated at 37°C for 3 h. The absorbance at 405 nm was measured with a Titertek Uniskan
reader. Sample NE concentrations were determined by
interpolation from a calibration line obtained with pure,
active-site titrated NE (kindly supplied by Dr David
Buttle, Strangeways Research Laboratory, Cambridge,
U.K.). Differences in NE activity were tested using
Student’s t-test.
Lactate dehydrogenase release. Cell culture supernatants were assayed for lactate dehydrogenase as previously described [ 151in order to measure the degree of any
cell death. Each supernatant (25 pl from cells preincubated with autologous plasma) was incubated with 1
ml of 0.05 mol/l phosphate buffer, pH 7.5 containing 70
pg of nicotinamide-adenine dinucleotide (reduced) and
Extracellular proteolysis by neutrophils
70 pg of sodium pyruvate. All reagents were obtained
from Sigma Chemical Co. The mixtures were incubated at
37°C and the decrease in absorbance at 340 nm was
recorded with a Perkin-Elmer 550 spectrophotometer.
Results were expressed as units of nicotinamide-adenine
dinucleotide (reduced)used min- ml- of sample.
'
37
from four subjects at a series of concentrations between
100 and 5 x lo3mol/l.
Cells were tested for viability after incubation with
endotoxin and cytokines by exclusion of Trypan Blue.
The effects of endotoxin and cytokines on FN digestion
were tested by analysis of variance and Students' f-test for
paired data.
Effects of endotoxin and cytokines
The effects of bacterial endotoxin and several cytokines on FN proteolysis were studied by incubating the
PMN from healthy, control subjects with 1251-FNas
above, in the presence of the materials described below.
Results were compared with control wells containing
medium only.
Bacterial lipopolysaccharide.Endotoxin (type 01 11:B4
from Escherichiu coli)was obtained from Sigma Chemical
Co., Poole, U.K. The cells from each of the six subjects
were incubated on the I2%FN with the endotoxin at a
concentrationof 1,2,4,6,8 and 10 pg/ml.
Recombinant tumour necrosis factor-a (TNF). This
was a gift from Dr G.R. Adolf of the Emst-Boehringer
Insitut Fur Arzneimittel-Forschung, Wien, Austria. The
TNF had a specific activity of 6 x lo6 units/mg. The
isolated PMN from six control subjects were adhered to
the FN with TNF added at concentrations of 1, 10, 100,
lo3, lo4 and lo5 units/ml. In a separate experiment the
effects of TNF on non-adherent PMN were investigated.
PMN from five volunteers were collected and treated with
TNF in two ways. First, the cells were adhered to FN and
TNF added at lo4 units/ml, as described above. The
control cells were incubated without TNF. Secondly,
PMN in suspension were preincubated with lo4 units of
TNF/ml for 30 min, the mixture being continually rotated
on a clynostat.
Interleukin-la (IL-1). Human recombinant IL-1 ( 10
ng/lOO units) was produced by The Genzyme Corporation, Boston, MA, U.S.A. and was obtained from KochLight Ltd, Haverhill, Suffolk, U.K. The effects of IL-1 on
the PMN from five subjects were studied at concentrations of 1.0,10,25,50 and 100 units/ml.
Leukotriene B, (LTB,). This was obtained from Orbit
Laboratories Ltd, Reading, U.K. The material was 99%
pure by h.p.1.c. The effects of LTB, on FN solubilization
by PMN from five subjects were studied at concentrations
of 10-lh, lo-',,
and
mol/l.
Interferon- y (IFN). IFN (Genzyme Corporation) was
obtained from Koch-Light Ltd and incubated with PMN
Effects of dexamethasone
Dexamethasone sodium phosphate was obtained from
Merck Sharpe and Dohme (Hoddesdon, Herts., U.K.).
The effect of dexamethasone on FN solubilization by
PMN from 10 subjects was tested at concentrations
between 10-I" and l o v 3 mol/l. Cell viability after 3 h
incubation at
mol/l was tested by exclusion of
Trypan Blue.
RESULTS
Patient studies
Fig. 1 shows the amount of FN proteolysed by 3 x lo5
PMN from each of the subjects studied. The patients' cells
preincubated with their own plasma solubilized significantly more (mean 3.2; SD 1.2 pg of FN) than those from
the control subjects which had been preincubated with
their own plasma (mean 2.1; SD 0.4 jig of F N P < 0.01).
Preincubation of the PMN from control subjects with
plasma from the patients with lung disease did not change
significantly the amount of FN solubilized (mean 2.2; SD
0.6 pg of FN). However, preincubation of PMN from the
patients with lung disease with control plasma significantly reduced (P<0.05) the amount of FN proteolysed
by these cells to a mean of 2.8 pg of FN; SD 0.88 pg of FN
(averagefall 10.4%).
The lactate dehydrogenase concentrations in the
supernatants collected after culturing the PMN from
control subjects on FN (mean 0.019; SD 0.016 units of
NADH used min- I ml- I ) were not significantly different
than those of the cells from patients with lung disease
(mean 0.016; SD 0.01 units of NADH min-' ml-l).
The NE concentrations in PMN supernatants are
shown in Fig. 2. The supernatants from the cells of the
patients with lung disease contained significantly more
Table 1. Effect of bacterial endotoxin on extracellulardigestion of FN by neutrophils
The results show the average amount of FN digested by 3 x lo5 cells in 3 h at six concentrations
of endotoxin, and by control cells to which no endotoxin was added. The increased FN digestion
achieved statistical significance (*P< 0.02, **P< 0.001) at concentrationsin excess of 6 pg/ml.
FN digested (pg/ml)
Endotoxin concn. (pg/ml) 0
Mean
SEM
2.67
0.95
1
2
4
6
8
10
2.99
0.92
2.98
0.91
3.07
0.80
3.28*
0.94
3.49"
0.84
3.65**
0.90
38
D. Burnett et al.
6.0
(P<0.002) NE (mean 0.97; SD 0.39 pg/ml) than the
supernatants from the control subjects’ cells (mean 0.60;
SD 0.16 pg/ml), although there was no significant correlation between FN digestion and NE concentrations in cell
supernatants.
y1
3
8
“0
0
4.0
0
0
3 :
0
”1
I
n
Control
-
Emphysema
Fig. 1. ( a )FN solubilization by neutrophils from healthy
control subjects, expressed as FN digested in 3 h by
3 x los cells. 0 , FN digested by cells preincubated with
their own (autologous)plasma; 0 , FN digested after the
cells were preincubated with plasma from the patients
with emphysema. ( b )FN digestion by neutrophils isolated
from patients with emphysema, expressed as FN digested
in 3 h by 3 X lo5. 0, FN digested by cells preincubated
with autologous plasma. 0 , FN digested by cells preincubated with plasma from the control subjects.
0
0
Effects of endotoxin and TNF
The addition of bacterial endotoxin to the PMN
cultured on ‘*’I-FN resulted in a significant (P<O.Ol,
analysis of variance) increased in FN digestion (Table 1).
Student’s f-test showed that the FN digestion was s i m i cantly higher in the presence of 6 (P<O.O2), 8 and 10
( P < O . O O l ) pg/ml than by the control cells.
Fig. 3 shows the effects of TNF on FN solubilizationby
PMN when the material was added to the adherent cells.
As with endotoxin there was a wide variation in the
degree of response by individuals’ cells, but TNF at concentrations in excess of 1O2 units/ml significantly
increased the amount of FN digested (P<O.O2) by all six
PMN samples with a maximum effect at lo4 units/ml
(P<O.Ol).Preincubation, with TNF, of PMN in suspension had no significant effect on subsequent FN digestion
(mean 0.78; SEM 0.17 yg of FN) compared with PMN
which were preincubated with medium alone (mean 0.58;
SEM 0.07 pg of FN). In contrast, when TNF was added to
the adherent PMN from the same five subjects the cells
digested significantly more ( P < O . O O l ) FN (mean 3.17;
SEM 0.459 yg of FN) than the untreated PMN (mean 0.96;
SEM 0.137 p g of FN; Fig. 4).
IL-1, LTB, and IFN had no significant effects on the
amount of FN solubilized by the PMN.
Exclusion of Trypan Blue indicated that cells remained
viable after incubation with cytokines and endotoxin.
0
0
6.0
1
5.0
-
4.0
-
0
00
‘
.-C
0
.
0
00
0.5
8
X
m
M
0
0
3
0
z
t;
Control
T T
Emphysema
Fig. 2. NE activity in the supernatants from neutrophils
cultured for 3 h on FN after preincubation with autologous plasma. Results are expressed as amount of
elastase/ml with reference to pure active-site titrated
enzyme.
3.0
-
2.0
I
1
I
o
I
I
I
I
I
I
1.0
10
102
103
104
105
TNF (units/ml)
Fig. 3. Effect of TNF on FN digestion by adherent
neutropMs. Results are means fSEM for six experiments.
39
Extracellular proteolysis by neutrophils
3.0-
4.01
4
T
2.0-
.n
2
.
-3
X
m
3
3
a
z:
1.0.
1
Cells
Cells + TNF
Adherent
Cells
Cells + TNF
Non-adherent
Fig. 4. Effect of TNF on FN digestion by PMN. The
hatched bars show the results (FN digested in 3 h by
3 x lo5 PMN, meansfsm) obtained when TNF ( lo4
units/ml) was added to the adherent cells. The left-hand
hatched bar shows the results from the control cells to
which TNF was not added. The open bars show the
results obtained when the PMN were preincubated in
suspension with TNF;the left-hand open bar shows the
results from control cells which were not preincubated
with TNF.
Effects of dexamethasone
The addition of dexamethasone to PMN cultured on
FN caused a significant dose-dependent reduction in
solubilization of the substrate (Fig. 5 ) , from a mean of
'11% inhibition at
mol/l (P<O.OOl) to 68% inhibition at
mol/l (P<O.OOl).The cells were found to
be 99% viable by Trypan Blue exclusion.
DISCUSSION
The results of the present study confirm our previous
observation that blood PMN from patients with chronic
bronchitis and emphysema can solubilize more extracellular protein than cells from healthy subjects of similar age
[ 131. Several studies have shown that PMN solubilize proteins on which the cells are cultured and have established
that NE is largely responsible for this process [4-71.
Campbell & Campbell [8] have shown, using immunofluorescence microscopy, that proteolysis occurs beneath
the cell as it moves across the substrate. The proteolysis
is therefore likely to be due to NE release in the pericellular area. The process of extracellular proteolysis by PMN
may be important for the movement of these cells from
the blood to inflamed tissues [ 161.
In the present study, we observed that the concentrations of NE in the PMN supernatants from patients with
0.
I
&lo
&-a
10-6
1
10-4
I
10-2
1
0
Dexamethasone concn. (M)
Fig. 5. Effect of dexamethasone on FN digestion by
neutrophils. The mean values ( ~ S E M ,n = 10) for conmol/l are
centrations of dexamethasone from 0 to
shown. The inhibition at
mol/l was significant at
P < 0.01; all other results were significant at P < 0.001.
lung disease were sipficantly higher than those from the
controls. These results suggest that the increased proteolysis of extracellular FN by cells from patients with lung
disease was due to increased extracellular release of the
contents of azurophil granules. Neutrophils contain about
0.2-5 pg of NE/106 cells [13] and during the 3 h incubation on FN the PMN had therefore released a large proportion (0.2-1.0 p g / l O h cells) of their total NE content.
Nevertheless, no significant correlation was observed
between the amount of NE released and the degree of FN
digestion. This suggests that total release of azurophil
granule enzymes do not reflect directly the degree of
enzyme activity in the pericellular area where extracellular proteolysis is thought to occur [8]. Clearly, other
factors may also be important, such as the degree of cell
spreading and adherence, which would influence the area
of close contact between cell and substrate. The concentrations of cytosolic lactate dehydrogenase were not significantly higher in the supernatants of cells which had
digested greater amounts of FN. This confirms that the
enhanced proteolysis by cells from subjects with lung
disease was not due to increased cell death and lysis in
culture.
The enhanced sensitivity to chemotactic factors
demonstrated by PMN from subjects with chronic
bronchitis and emphysema [12, 131 may lead to an inappropriately excessive recruitment of neutrophils to the
40
D. Burnett et al.
lungs. Furthermore, results in vitro suggest that these cells
digest large amounts of extracellular protein as they migrate through tissues [ 161. This process would be
exacerbated by the presence of activating factors, since
even the ‘hyperactive’ PMN from patients with lung
disease have the potential to be stimulated further [ 131.
In the present study we have considered the possibility
that the greater proteolysis by PMN from patients with
chronic bronchitis and emphysema may be due to activation of the cells by factors released as a result of lung
inflammation. Several factors, such as bacterial endotoxin,
TNF and INF have been shown to increase superoxide
production, lysosomal enzyme release and phagocytic
activity of PMN [17-191. We found that endotoxin and
TNF increased extracellular proteolysis by the PMN,
although the degree of response by PMN from different
individuals varied greatly and the effects of endotoxin
were only observed at high concentrations. Others have
observed a wide range of PMN response to TNF with
regard to superoxide production [ 181. In contrast, IL- 1,
IFN and LTB, had no significant effects in our system.
The absence of stimulation by IFN was surprising in view
of previous reports [18]. It may be that NE release and
extracellular proteolysis are features of neutrophil behaviour that are not affected by this cytokine.
Alternatively, the results obtained in studies of PMN
activation can, for technical reasons, be ambiguous. For
instance, the method of neutrophil isolation or the
presence of trace amounts of endotoxin can prime these
cells in vifro [ 171. In the present study, Percoll gradients
were used to isolate the neutrophils. This procedure has
been shown not to activate PMN in vitro provided that
reagents are free of endotoxin. We confirmed that our reagents contained levels of endotoxin that were unlikely to
activate the cells during isolation and, as in a previous
study [13], we confirmed that the cells did not exhibit
changes in shape that are characteristic of activation [ 171.
IL-1 at concentrations in excess of 1 ng/ml has been
shown to induce hydrogen peroxide production and
specific granule release, but not azurophil granule release
by neutrophils [20].The absence of any effect of IL-1 on
FN digestion may reflect its inability to cause the release
of azurophil granule contents, including NE. Nevertheless,
the highest concentration of IL-1 used in the present study
was 1 ng/ml due to the limited amounts in the available
preparations. It is possible that higher concentrations of
IL-1 may yet be shown to influence extracellular proteolysis by PMN, although the physiological significance of
such high levels may be small.
It has been reported [21] that TNF can trigger hydrogen peroxide release by adherent PMN but not by cells in
suspension. Similarly, we found that if PMN were preincubated in suspension with TNF, extracellular proteolysis was not increased, in contrast to when the TNF was
added to the cells after adherence to FN. The stimulatory
effect of TNF on extracellular proteolysis therefore also
appears to be dependent on cell adherence. Preincubation
of neutrophils from control subjects with plasma from the
emphysema patients had no significant effect on proteolysis of the FN substrate. In view of the apparent depen-
dence on cell adherence for stimulation by factors such as
TNF, any activation of PMN in the circulation appears
unlikely. Nevertheless, these results do not preclude the
possibility that activating factors such as TNF are present
in the blood of subjects with lung disease. It is possible
that cell activation occurs while the cells are in contact
with the stroma of the bone marrow. It is not clear at
present whether full adherence is required for PMN
stimulation by TNF and partial adherence or contact with
a suitable substrate might be sufficient. It this were the
case, it might be possible for PMN marginating within the
pulmonary vasculature to be stimulated before re-entering the peripheral circulation. Clearly further studies will
be required to answer these questions.
The observation that preincubation of PMN from the
emphysema patients with plasma from control subjects
caused a reduction in FN digestion suggests that the
blood normally contains suppressive factors which may
be deficient in lung disease. Nevertheless, this suppression
was modest in comparison with the degree of increased
FN digestion by the cells from patients with lung disease.
The potential to inhibit extracellular proteolysis by
neutrophils was demonstrated, however, by the addition
of dexamethasone to the cell cultures. A significant degree
of FN proteolysis (11%) was seen with only 10- lo mol/l
dexamethasone. We have shown previously that corticosteroid treatment of patients with chronic bronchitis
resulted in an increase in the NE inhibitory capacity of the
lung secretions [ 141. This effect was not due to increased
secretion concentrations of a,-antitrypsin [22]. The
results of the present study suggest that the increased NE
inhibitory capacity of sputum after corticosteroid therapy
might be explained by a reduction in NE release from
neutrophils, which would leave more a ,-antitrypsin
functional as an inhibitor in the secretions.
In conclusion, the greater extracellular proteolytic
activity by PMN from subjects with chronic obstructive
lung disease might contribute to the pathological lesions
causing emphysema. The reasons for this altered cell
behaviour remain unresolved, but might result from
increased stimulation of cells by factors released because
of lung inflammation and reduced suppressive factors in
the blood.
ACKNOWLEDGMENTS
D.B. is a British Lung Foundation/British Oxygen
Company Centenniel Fellow. We are greateful for support
from the Chest, Heart and Stroke Association, the TSB
Foundation and the West Midlands Regional Health
Authority.
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