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Inhibition of neutrophil-mediated degradation of isolated basement membrane collagen by nonsteroidal antiinflammatory drugs that inhibit degranulation.

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INHIBITION OF NEUTROPHIL-MEDIATED
DEGRADATION OF ISOLATED
BASEMENT MEMBRANE COLLAGEN
BY NONSTEROlDAL ANTIINFLAMMATORY DRUGS
THAT INHIBIT DEGRANULATION
TERESA M. NEAL, MARGRET C. M. VISSERS, and CHRISTINE C. WINTERBOURN
The ability of nonsteroidal antiinflammatory
drugs to inhibit neutrophil-mediated degradation of
type IV collagen in an in vitro tissue injury model using
glomerular basement membrane (GBM) containing immune complexes was investigated. Auranofin (2.5-10
p.40, phenylbutazone (50-250 /..
sulfasalazine
&I),
(2501,000 Eun), and 4-bromophenacyl bromide (5-20 luM)
each inhibited up to 70% of the collagen degradation, in
parallel with almost complete inhibition of the release of
azurophil and specific granule enzymes. These drugs
had much less an effect on gelatinase release. Indomethacin and the antimalarials, which inhibited the
neutrophil oxidative burst but not degranulation, had
little effect on GBM collagen degradation. Our results
do not necessarily imply that inhibition of neutrophilmediated degradation of connective tissue is relevant to
the action of nonsteroidal antiinflammatory drugs in
vivo; however, using the GBM model system, we have
shown that when a drug inhibits granule enzyme release, there is an associated decrease in collagen degradation, whereas inhibition of the oxidative burst has
relatively little effect.
Polymorphonuclear leukocytes (neutrophils)
are prominent at sites of inflammation and are implicated as a cause of tissue injury ( 1 4 ) . These cells
From the Pathology Department, Christchurch School of
Medicine, Chnstchurch, New Zealand.
Supported by a grant from the Medical Research Council of
New Zealand.
Teresa M. Neal, MSc; Margret C. M. Vissers, PhD;
Christine C. Winterbourn, PhD.
Address reprint requests to Dr. C. C. Winterbourn, Pathology
Department, Christchurch School of Medicine, Chnstchurch, NZ.
Submitted for publication August 11, 1986; accepted in
revised form January 28, 1987.
Arthritis and Rheumatism, Vol. 30, No. 8 (August 1987)
infiltrate the cartilage of arthritic joints (2,3) and
adhere to the glornerular basement membrane (GBM)
in glomerulonephritis (4). Neutrophils are stimulated
by a variety of inflammatory mediators to generate
reactive oxygen species and release neutral proteinases, both of which can damage tissue components
(5). A number of nonsteroidal antiinflammatory drugs
fNSAIDs) inhibit neutrophil superoxide production
and degranulation (6-11), and it has been proposed
that this is one mechanism whereby the drugs could
halt the disease process.
The present study was undertaken to determine
whether inhibition of these responses correspondingly
decreases the ability of neutrophils to degrade
extracellular matrix proteins. The effects of each drug
can depend on the neutrophil stimulant (7-11); for
example, a number of drugs which, at low concentrations, inhibit responses to the chemotactic peptide,
fMet-Leu-Phe, are ineffective when the cells are stimulated with immune complexes (ICs), and others are
effective only at higher concentrations (1 1).
Immune complexes are considered to be a
major stimulus in chronic inflammation. Their ability
to induce neutrophil-mediated tissue damage has previously been studied in an in vitro model that consisted
of isolated GBM impregnated with IgG aggregates
(12). The IC within the membrane cause neutrophils to
release oxidants and granule enzymes on the surface
and to degrade the GBM protein (12-16). The enzymes
responsible for degradation of type 1V collagen are
elastase, gelatinase, and cathepsin G, which contribute 60%, 30%, and lo%, respectively (15,16). We have
examined the effects of NSAIDs in this model system.
This has enabled us to determine whether the drugs
have similar effects on cells stimulated by free ICs and
BASEMENT MEMBRANE DEGRADATION
by surface-bound ICs, and to directly assess how
modulation of neutrophil responses affects collagen
degradation.
We have investigated those drugs previously
shown to inhibit neutrophil responses to ICs in suspension ( 1 l), namely, auranofin, sulfasalazine, indomethacin, phenylbutazone, and the antimalarials, chloroquine, quinacrine, and primaquine. The phospholipase
A2 inhibitor, 4-bromophenacyl bromide, was also
tested.
MATERIALS AND METHODS
Materials. Ficoll 400 and QAE Sephadex A-50 were
obtained from Pharmacia (Uppsala, Sweden), Hypaque from
Sterling Drug (New York, NY), and o-tolidine from Merck
AG (Darmstadt, FRG). Auranofin was a gift from Smith
Kline & French (Sydney, Australia), and sulfasalazine was
from Pharmacia (North Ryde, Australia). Other chemicals
were obtained from Sigma (St. Louis, MO).
Drug solutions were always freshly prepared.
Auranofin was dissolved in ethanol; chloroquine and
quinacrine were dissolved in water. The other drugs were
dissolved in dimethylsulfoxide to a final solvent concentration of <0.1%. Control experiments showed that this concentration of solvent had no effect.
IgG was purified from human plasma by precipitation
with ammonium sulfate and ion-exchange chromatography
on DEAE-Sephadex (17).
Neutrophils. Human neutrophils were isolated from
the blood of healthy donors by Ficoll-Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of contaminating red cells (18). Cell suspensions regularly contained
95-96% neutrophils and 3-5% eosinophils, and were >98%
viable, as assessed by trypan blue exclusion. The cells were
suspended at 107/ml.Incubations were carried out in 10 mM
phosphate buffered saline, pH 7.4, supplemented with 1 mM
CaC12, 0.5 mM MgC12, and 1 mg/ml glucose.
GBM containing IgG aggregates. Intact glomeruli
were prepared (19) from histoiogicaIIy normai human kidneys obtained at autopsy, and GBM was extracted by
detergent solubilization and DNase treatment (20). To impregnate it with IgG aggregates, GBM was soaked in 10
mgirnl purified human IgG, heated at 63"C, and washed with
incubation buffer as previously described (12).
Neutrophil interaction with GBM. Neutrophils (6 x
106in 0.6 ml) were preincubated for 5 minutes with each drug
(except auranofin, which required 30 minutes), then were
added to 1.2 mg of GBM containing IgG aggregates. After 2
hours at 37"C, the cells and insoluble GBM were pelleted by
centrifugation at 1,OOOg for 5 minutes. The extent of neutrophi1 degranulation was determined by measuring granule
enzyme markers released into the supernatant. Lysozyrne
was measured using Micrococcus lysodeikticus (21), and
P-glucuronidase and myeloperoxidase were measured with
o-tolidine (22). Gelatinase activity was measured against
heat-denatured collagen (gelatin) (23). The collagen was first
labeled by oxidation with formaldehyde and treatment with
3H-NaBH4(24). Lactate dehydrogenase was monitored as an
909
indicator of cell viability (25). The respiratory burst was
monitored by measuring oxygen consumption with a Clarktype oxygen electrode and a YSI bioiogical oxygen meter.
Neutrophils (2.5 ml) were preincubated at 37°C with each
drug, 5 mg of GBM was added, and the initial linear rate of
oxygen uptake (over 5 minutes) was calculated.
Degradation of the GBM was measured by determining the release of soluble collagen fragments (12). A portion
of the supernatant was hydrolyzed in 6N HCl, and soluble
hydroxyproline was determined by reaction with chloramine
T and p-dimethylaminobenzaldehyde (26).
RESULTS
Drug effects on oxygen consumption and degranulation. Neutrophils were stimulated by IgG aggregates trapped within GBM to release the granule
enzyme markers lysozyme, P-glucuronidase, myeloperoxidase, and gelatinase (Table 1). The cells also
exhibited a marked increase in oxygen consumption
(Table 1). Previous studies (12,13) have shown that the
cells adhere to the membrane, and the responses do
not occur with GBM alone or with nonaggregated IgG.
The effects of the NSAIDs on oxygen consumption and granule enzyme release are shown in Figures
Table 1. Oxygen uptake, granule enzyme release, and collagen
degradation by neutrophils stimulated by glomerular basement
membrane (GBM) containing IgG aggregates*
UnstimuKd
neutrophilst
Oxygen uptake
0.11 5
(nmoleslminute/lO6 cells)
p-glucuronidase release
1.9
(units/1O6cells)
0.04 5
Myeloperoxidase release
(units/W cells)
Lysozyme release
0.005 2
(units/106 cells)
Gelatinase release
2.7 C _
(% gelatin solubilized/lOb
cells under assay
conditions)
Hydroxyproline solubilized
0.07 5
(pg/106 cells)
0.06
* 0.5
Neutrophils +
basement membrane
1.9 2 0.5
0.02
* 5.7
0.23 * 0.14
0.003
0.020 5 0.006
2.7
0.04
8.8
40
t_
5
1.13
?
0.63
* Reaction conditions are described in Materials and Methods.
Results are mean 2 SD for at least 20 assays. A unit of pglucuronidase corresponds to 1 pg of phenolphthalein releasedh8
hours, and 1 unit of myeloperoxidase or lysozyme corresponds to an
absorbance change of liminute. Total release, compared with sonicated cells, was -8-10% for p-glucuronidase and myeloperoxidase,
30% for lysozyme, and 30% for gelatinase.
t Enzyme release and oxygen uptake were measured on unstimulated
cells incubated at 37°C for 2 hours with no GBM. These values do not
differ significantly from measurements on cells in the presence of
G B M without IgC aggregates (see refs. 12 and 13). Hydroxyproline
was measured after adding neutrophils to GBM containing no IgG
aggregates.
NEAL ET AL
910
PI
a-yyiEqm
e':Lr
Oxygen consumption and
enzyme release
A.,
v
"
9
ae
5
Oxygen consumption and
enzyme release
Basement membrane
degradation
Basement membrane
degradation
---
r. ..
..
0
0
500
XKK)
m \
~
+----
0
500
too0
0
--1
loo0
500
0
lndomethacin ( @ M )
Chloroquine ( p M)
Phenylbutazone ( p M )
Quinacrine ( MI
500
1ooo
wLh
d*;b;
E:h
h*l
Primaquine (,,MI
Auranofin ( pM)
Ve
s
s
o
V
s
8
...
'\i
0
500
lo00
0
500
moo
Sulfasalazine ip M)
0
10
20
0
m
20
4-Erornophenacyl bromide ( pM1
Figure 1. Effects of antiinflammatory drugs on neutrophil oxygen uptake and granule enzyme release (A-H) and on collagen degradation (a-h)
when neutrophils were added to glomerular basement membrane containing IgG aggregates. Incubation conditions are described in Materials
and Methods. For control values in the absence of drug, see Table 1. Each point represents the mean 2 SD of 2 observations ( 0 2 uptake) and
3 4 observations for the other parameters. 0------0
= oxygen uptake; 0
-0
= lysozyme release; 0-0
= release of azurophil marker
(p-glucuronidase for indomethacin and primaquine, myeloperoxidase for the other drugs).
1A-H. All drugs tested inhibited oxygen consumption,
although the concentrations required and the extent of
inhibition differed. Whereas 10 pM auranofin (Figure
1C) or 20 p M 4-bromophenacyl bromide (Figure 1H)
gave complete inhibition, 1,000 pM indomethacin
(Figure 1A) or sulfasalazine (Figure 1D) was required
to inhibit the respiratory burst by -70%.
Release of the azurophii and specific granule
markers, p-glucuronidase or myeloperoxidase and
lysozyme, was strongly inhibited by low concentra-
91 1
BASEMENT MEMBRANE DEGRADATION
Table 2. Effect of nonsteroidal antiinflammatory drugs on
release of gelatinase from neutrophils stimulated by glomerular
basement membrane containing IgG aggregates*
Concentration
D w
(CLM)
Indomethacin
Phenylbutazone
Auranofin
Sulfasalazine
Chloroquine
Quinacrine
Primaquine
4-bromophenacyl bromide
1,000
250
10
1,000
1,000
500
1,000
10
Gelatinase release
(% of control)t
96 2
32 5
80 5
69 2
109 2
185
?
44
92
2
5
10
30
7
16
16
14
40
9
* Results are mean 5 SD of 3-6 estimations.
t Control values are given in Table 1.
tions of auranofin (Figure 1C) and 4-bromophenacyl
bromide (Figure lH), and by higher concentrations of
sulfasalazine (Figure 1D ) and phenylbutazone (Figure
1B). Indomethacin (Figure 1A) and primaquine (Figure
IG) had no effect, and chloroquine and quinacrine
stimulated enzyme release (Figures 1E and F). This
was not due to cell lysis, because release of lactate
dehydrogenase, a cytoplasmic marker, was the same
as was seen with unstimulated cells. Neutrophils have
been shown to oxidatively inactivate their own granule
enzymes when stimulated on GBM (16). Since
chloroquine and primaquine markedly inhibited oxidant production, the increased enzyme activities measured in the presence of these drugs were probably due
to decreased oxidative inactivation. In general, there
was slightly greater inhibition of release of the
azurophil marker (myeloperoxidase or P-glucuronidase) than of lysozyme, which is present in azurophil
and specific granules; however, the extent of inhibition
of lysozyme release is sufficient to imply that the
inhibitory drugs affect release of both granules.
Gelatinase contributes -30% to the GBM degradation by neutrophils (15,16). It is thought to reside
in a separate secretory granule, the C-particle (27),
although a recent report (28) suggests a specific granule origin. In contrast to the other enzymes measured,
gelatinase release was not greatly altered in the presence of most of the drugs (Table 2). Inhibition was
seen only with phenylbutazone, primaquine, and
sulfasalazine, and then only at the top of the concentration range. This is consistent with our findings with
free ICs (11). That auranofin and sulfasalazine, at
concentrations sufficient to prevent 90% of lysozyme
release, inhibited gelatinase release by only 20-30%
suggests that the 2 enzymes are under separate con-
trol, and supports the proposal that they are in different granules (27).
Drug effects on GBM degradation. Neutrophils
added to GBM caused degradation of the membrane
collagen to soluble hydroxyproline-containing fragments (Table I), as reported previously (12). Figures la-h show that the drugs which inhibited
neutrophil enzyme release also markedly decreased
hydroxyproline solubilization. Thus, indomethacin
(Figure la), which had only a slight effect on
degranulation, did not significantly alter degradation,
whereas phenylbutazone (Figure lb), auranofin (Figure 1c), sulfasalazine (Figure 1d), and 4-bromophenacyl bromide (Figure lh), which were potent
inhibitors of enzyme release, also decreased GBM
degradation, with a maximum of -70% inhibition.
Chloroquine and quinacrine (Figures le and 0 ,
which inhibited oxygen uptake only, did not affect
degradation. This was despite increased granule enzyme release, which might be expected to give greater
degradation. Primaquine, which did not affect enzyme
release, although it inhibited oxygen uptake, decreased hydroxyproline solubilization by up to 30%.
Since a number of NSAIDs bind to albumin, the
effects of 3% albumin on enzyme release and GBM
degradation were examined. Conditions were the same
as described in Figure 1. The effects of sulfasalazine
(250 p M and 1,000 pM) were comparable in the
presence and absence of albumin. Phenylbutazone
(250 pkf) in the presence of albumin, however, did not
affect lysozyme release, and caused decreases of only
25% in myeloperoxidase release and 20% in hydroxyproline solubilization. Auranofin (5 pM) was also less
effective, with no inhibition of lysozyme release and
40% and 10% decrease in myeloperoxidase and hydroxyproline release, respectively.
DISCUSSION
A growing number of NSAIDs have been
shown to affect neutrophil function. When release of
oxidants or degradative enzymes is inhibited, an associated decrease in tissue injury would be expected, but
until now, this has not been directly demonstrated.
Using a model system in which ICs are trapped within
a GBM matrix, we found that inhibition of specific and
azurophil granule release is associated with a corresponding decrease in degradation of the GBM
collagen. Since most of the degradation is due to
elastase (15,16), which is contained in azurophil granules, there is a logical progression from inhibition of
912
enzyme release to inhibition of digestion. When release of azurophil granules was completely inhibited,
e.g., by auranofin, sulfasalazine, or 4-bromophenacyl
bromide, there was still -30% of normal collagen
degradation. This corresponds to the expected contribution of gelatinase (15,16), and since these drugs had
only a small effect on gelatinase release, it probably
accounts for the residual degradation.
Auranofin, phenylbutazone, sulfasalazine, and
the phospholipase A2 inhibitor, 4-bromophenacyl bromide, each substantially inhibited GBM degradation.
The effects of the drugs on neutrophil O2 uptake and
degranulation observed in this study parallel their
effects on neutrophils stimulated by free ICs (1 1). The
dose responses are similar in each case, and the only
noteworthy difference is that indomethacin and
primaquine gave more inhibition of degradation with
free ICs than with GBM-associated ICs. Thus, it
appears that drug responses are not altered by surface
attachment of ICs and “frustrated phagocytosis,” and
extrapolation can be made from one system to the
other. Piroxicam, mefenamic acid, ibuprofen, and
penicillamine, which do not inhibit IC-induced degranulation (1 I), would not, therefore, be expected to
inhibit collagen digestion.
Apart from ICs, other inflammatory mediators
can also induce neutrophil enzyme release. The effects
of NSAIDs depend on how the cells are stimulated;
responses to ICs are generally more resistant to inhibition than responses to stimuli such as Wet-Leu-Phe
(8-11). The extent to which drugs could modulate
neutrophil-mediated extracellular matrix degradation,
therefore, should depend on which stimulant is dominant. Drugs such as indomethacin, piroxicam, and
mefenamic acid, which are good inhibitors of responses to met-Leu-Phe (1 I), may inhibit degradation
induced by stimuli other than ICs, and sulfasalazine
and phenylbutazone may then be effective at lower
concentrations.
Inhibition of the neutrophil respiratory burst,
but not of degradation, was found to have little effect
on GBM collagen degradation. This is consistent with
the results of a recent study (16) showing that
neutrophil oxidants both increase the susceptibility of
GBM to proteolysis, and inactivate the enzymes released from the cells. As a result of these opposing
effects, oxidants make little contribution to collagen
degradation on a per-cell basis. Our findings that
chloroquine and quinacrine enhance enzyme activity
(i.e., they decrease oxidative inactivation) without
increasing digestion are compatible with this explana-
NEAL ET AL
tion. The slight decrease in digestion without a decrease in released enzyme activity, as seen with
primaquine and indomethacin, could also be due to
inhibition of the oxidative burst decreasing the
proteolytic susceptibility of the GBM.
Although the role of neutrophils in inflammatory tissue injury is uncertain, they have been seen
invading arthritic cartilage (2,3) and adjacent to GBM
in nephrotoxic nephritis (4). ICs within the matrix,
which induce neutrophils to adhere and release enzymes and oxidants at the area of contact (12,13), are
likely to be a major stimulus for matrix degradation.
Because a number of NSAIDs do not affect responses
to ICs, it is unlikely that inhibition of matrix protein
degradation is a universal feature of NSAlD action.
The relatively high concentration requirement for
those drugs which do inhibit the process, and the
decreased effects in the presence of albumin, also
make this role questionable. It is also noteworthy that
gelatinase release was relatively insensitive to the
drugs, so its action (potentially against types IV and V
collagen, as well as various glycoproteins) is not
readily preventable. Our findings in the basement
membrane model system have shown, however, that a
link can be made between inhibition of neutrophil
degranulation and matrix protein degradation, and the
model provides a means to assess whether antiinflammatory agents can modulate the process.
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