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Enzyme release from human leukocytes and degradation of cartilage matrix effects of antirheumatic drugs.

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Enzyme Release from Human Leukocytes
and Degradation of Cartilage Matrix
Effects of Antirheumatic Drugs
R. J. Perper and A.
L. Oronsky
Various antirheumatic drugs were tested in a newly described model of
cartilage degradation-induced by the nonphagocytic release of neutral
protease from viable human leukocytes in the presence of aggregated
human lgG. Steroidal and nonsteroidal antiinflammatory drugs inhibited
the enzyme release process but not neutral protease activity, whereas
goid and chloroquine inhibited the enzyme activity without affecting the
release process. Several other drugs, known not to have antiinflammatory
activity, were inactive. Neutral protease appears to be preformed in
neutrophils and not induced by de novo synthesis during phagocytosis.
The concentrations at which the antirheumatic drugs were active in
vitro correlated with blood levels usually achieved in man. It Is suggested
that the activity of compounds in the model system described might predict their effect in vivo in rheumatoid arthritis.
An important facet of the pathology of
various inflammatory disease states is the
influx of a variety of circulating leukocytes
to the area of inflammation. T h e polymorphonuclear leukocyte may play a uitical role in the development of the inflammatory response since phagocytosis of particulate matter, such as inert particles or
antigenlantibody complexes, results in the
release of various enzymes from these cells
From the Research Department, Pharmaceutical
Division Ciba-Geigy Corporation, Ardsley, New York
10502.
R J PERPER, DVM, PhD: Adjunct Associate Professor Pathology, New York Medical College, New
York, New York; A L ORONSKY, PhD: Adjunct Assistant Professor Pathology, New York Medical College, New York, New York.
Address reprint requests to Dr R J Perper, CibaGeigy Corporation, Ardsley, New York 10502.
Submitted for publication July 10. 1973; accepted
September 24, 1973.
(1,2). The relevance of this process to the
inflammatory response has been questioned
since in experiments heretofore reported
the released enzymes were active only at a
low pH. It has been shown however that
extracts of human leukocytes contain enzymes capable of reducing the viscosity of
protein polysaccharide solutions at neutral
pH (3) and we have demonstrated that
these enzymes, also under physiologic conditions, can degrade the mucopolysaccharide matrix of rabbit ear cartilage (4).
Of interest in this regard is the finding that
neutral protease activity is present in human but not in rabbit or guinea pig leukocytes. A new in vitro model has been described recently ( 5 ) in which viable human
peripheral leukocytes release neutral protease(s) during the process of phagocytosis,
which degrades the matrix of cartilage
coated with aggregated human gamma
Arthritis and Rheumatism, Vol. 17, No. 1 (January-February 1974)
47
PERPER AND ORONSKY
globulin. The present study deals with the
effects of various antiinflammatory drugs
on the nonphagocytic release of granuleassociated netural protease from human
leukocytes, utilizing the %-labeled rabbit
ear cartilage model for estimating protease
activity. In addition the drugs were tested
for their capacity to directly inhibit protease activity.
MATERIALS AND METHODS
Nas"S04 (1000 mCi/mM) was obtained from New
England Nuclear Company, Boston, Massachusetts:
Hanks' Balanced Salt Solution (BSS) (without
phenol red) was purchased from Microbiological
Associates, Bethesda, Maryland. Insta-Gel Emulsifier (Packard Instruments, Downers Grove, Illinois)
and Dextran 70 in normal saline (Cutter Laboratories, Berkeley, California) were also used. T h e
following drugs were utilized in the present experiments: gold sodium thiomalate (Merck, Sharpe and
Dohme), acetylsalicylic acid (Sigma), oxyphenbutazone and phenylbutazone (Geigy Pharmaceuticals),
indomethacin (Merck, Sharpe and Dohme), chloroquine phosphate (Sigma), hydrocortisone hemisuccinate (Sigma), chlorpromazine (Smith, Kline and
French), dextropropoxyphene (Lilly), pyribenzamine
maleate (Ciba Pharmaceuticals), chlorothiazide
(Merck, Sharpe and Dohme), codeine sulfate (Ciba
Pharmaceuticals), puromycin hydrochloride (Sigma),
chloramphenicol (Parke-Davis). Acetylsalicylic acid,
oxyphenbutazone, phenylbutazone, and indomethacin were solubilized in exact molar equivalents of
NaOH. All other compounds were soluble in aqueous media. Human IgG (Fraction 11) was obtained
from Miles Laboratories, Kankakee, Illinois.
steel cork borer. The discs were washed thoroughly
in large volumes of cold saline. The cartilage was
blotted dry and either used immediately or stored
at -70% for up to 4 weeks. Individual discs
weighed 3.8-4.2 mg.
Preparation of Human Leukocyte
Suspensions
Human peripheral blood leukocyteswere obtained
from healthy volunteers who were seronegative for
rheumatoid factor. One hundred milliters of heparinized blood was obtained by venopuncture, and
erythrocytes were sedimented in the presence of 6%
dextran and glucose at room temperature for 90
minutes. The leukocyte-rich layer was withdrawn
and centrifuged at 120 X g for 10 minutes at 4OC.
The cell sediment was resuspended in 20 ml of cold
Hanks' BSS (pH 7.4) containing 1% glucose ("/v).
After another centrifugation the leukocytes were
resuspended in Hanks' BSS containing 1% glucose
at a concentration of 3.3 X loe cells per milliliter.
This suspension contained about 50y0 neutrophils,
4070-5070 lymphocytes with 2%-50/, monocytes and
10/,-20/, eosinophils. Ninety-nine percent of these
cells excluded trypan blue dye, using standard dye
exclusion techniques.
In one experiment whole blood diluted 1:4 with
Hanks' BSS solution was layered over a FicollHypaque cushion and centrifuged at 400 X g for
20 minutes, according to the technique described
previously (6). T h e mononuclear cell layer was removed (98% lymphocytes, 2% monocytes) and these
cells were washed twice in Hanks' BSS solution and
suspended in a final concentration of 3.3 X lo8
cells per milliliter for preparation of lysates.
Opsonization of Cartilage with
Human Aggregated IgG
Preparation of Cartilage
The preparation of cartilage has been described
previously (4). New Zealand white rabbits (1 kg,
male, HARE) were injected intravenously with 750
pCi of Na2=S0418 hours prior to sacrifice by cervical dislocation. All subsequent steps were performed
at 4°C. Both ears were amputated and stripped of
all adhering layers of epithelial tissue and fascia.
Only the center sections of the ear cartilage were
used for experimental purposes. These sections were
soaked in cold sterile saline, blotted dry and 3-mm
diameter discs were removed with a small stainless
40
Human IgG (50 mg) was dissolved in 6 ml of
phosphate buffered saline and heated for 20 minutes at 63°C. A second addition of 100 mg of IgG
was added to the tube maintained at 63°C. After
20 minutes the aggregated IgG was centrifuged at
8000 X g for 15 minutes at 4OC. The sediment was
washed, resuspended in 2.5 ml of Hanks' BSS, and
stored at 4°C. T h e cartilage discs were opsonized
by incubation with aggregated IgG suspension at
37°C. After 15 minutes the discs were removed,
washed in Hanks' BSS, and placed in the reaction
vessel.
Arthritis and Rheumatism, Vol. 17, No. 1 (January-February 1974)
EFFECT OF ANTIRHEUMATIC DRUGS
Release of 35Sand p-Glucuronidase
from labeled rabbit ear cartilage during the
first 5 hours of incubation in Hanks' BSS
Opsonized cartilage (5 pieces
20 mg) was in- at pH 7.4 (4). I n the presence of human
cubated alone, with 5 X loa cells or with 5 X loe leukocytes 40y0-5070 of the total radiocells plus drug in 1.5 ml of Hanks' BSS at p H 7.4
activity was released when the cartilage was
(37°C) for 5 hours. In the preincubation experiments 5 X lo" cells were incubated with the drug coated with human aggregated IgG (5).
or the vehicle for 1 hour only a t 37°C. The carti- These experiments were repeated, and the
lage was then added to this vessel and incubation data in Table 1 confirm the previous findwas continued for 5 hours. The incubation media ings since 792 cpm (5y0) of the total s%
contained penicillin (lOO,/ml) and streptomycin
were released into the cell-free incubation
(lOO,.t/ml). After incubation 0.5-ml aliquots of the
medium were removed and added to 10 ml of media after 5 hours incubation of 20 mg of
Insta-Gel. T h e samples were analyzed for "S with 3JS-labeled cartilage containing a total of
a Packard Tricarb liquid scintillation spectrometer. approximately 16,000 cpm. Release of 35S
Total Y3 in the cartilage was determined by dis- was increased ten fold in the presence of 5
solving the cartilage in 2.0 ml of hot concentrated
x lo6 human leukocytes (7893 cpm). I n the
HCl, and counting 0.1-ml aliquots in 10 ml of
presence
of a pure lymphocyte suspension
Insta-Gel.
(5
x
lo6
mononuclear cells) only 960 cpm
Data in the text are the average of triplicate determinations expressed as the fraction of total "S
were released.
cpm in the sample, released after subtracting backColchicine, present in the incubation
ground radiation (20-25 cpm). Efficiency of "S
media
at a concentration of lO-dM, 10-5M,
counting was approximately 70%. p-glucuronidase
or
10-6M,
inhibited leukocyte-induced 35S
(p-G) expressed as optical density (OD) units was
release
by
58%,
45%, and 4%, respectively
determined by the method of Gianetto and DeDuve
(Table
1).
Determinations
of p-G activity
(7).
in the media were used to estimate the
amount of lysosomal enzymes released by
Preparation of Lysates of Total Granule
human leukocytes during the abortive
Fraction from Human Leukocytes
phagocytic process. Colchicine at concenHuman leukocytes prepared as described above
were adjusted to a final concentration of either 6.0 trations of lO-dM, lO-5M, and 10-6M inhibited p-G release 67y0, SO%, and SOY0,
X lo' or 1.2 X 108 cells per milliliter. The total
granule fraction was isolated as previously de- respectively. Inhibition of 35S release ocscribed (4). T h e isolated granules were suspended curred in the presence of 10-4M and
in Hanks' BSS to a volume equivalent to that in
10-"M concentrations of phenylbutazone
which the leukocytes were suspended just prior to
(39Y0
and 20%), oxyphenbutazone (49y0
cell disruption. The granules were then subjected
and
SO%),
and indomethacin (49% and
to 10 freeze-thaw cycles, using a mixture of dry iceacetone, and centrifuged at 15,000 X g for 15 min17y0). All of these agents were ineffective at
utes at 4%. Supernatants were used immediately.
10-6M. T h e inhibition of P-G release was
The human lymphocyte suspension was subjected roughly parallel to changes in 3 5 s release.
to a similar freeze-thaw procedure and then hoAspirin was effective only at 10-SM [21y0
mogenized at 4OC with a Teflon pestle for 3 mininhibition
of 35S release and 24y0 inhibiutes. The final preparation contained no intact
cells, as judged by phase-contrast microscopy.
tion of p-G release (Table l)]. There was
no significant effect of aspirin a t lO-"M on
either
release or p-G release when comRESULTS
pared to controls. Cyclophosphamide a t
I t was previously shown that there is ap- 10WSM and 10-4M did not inhibit 35Sreproximately a 10% autolytic release of 35s lease and increased p-G release by approxi-
@-G) from Ear Cartilage
Arthritis and Rheumatism, Vol. 17, No. 1 (Januaty-February 1974)
49
PERPER AND ORONSKY
Table 1. Effect of Drugs on Nonphagocytic Enzyme Release
from 5 X 10' Human Leukocytes
Concentration
Drug
Cartilage alone
Cartilage 5 x loe
lymphocytes
Cartilage 5 X 10'
leukocytes
Colchicine
(MI
792
+
+
Aspirin
Oxyphenbutazone
lndornethacin
Chloroquine SO,
Gold Na thiornalate
Hydrocortisone
hernisuccinate
Cyclophosphamide
Inhibition
=s (%)*
* 130t
10"
1 o4
1 0-e
1 o4
10-4
1 Od
1 010-4
1 o4
104
10-4
1 o=
1010-4
1 o=
1 010-4
1 o4
1o4
10-7
'
0
1
1 o4
1 o-a
1 o4
7.893 f 678
3,774 f 27
5,430
7,590
6,383 541
7,010f 31 1
5,130 476
6,450
7,680
4,094
5,730
7,350
4,422
6,690
7,920
8,911 f 953
10,800
7,116
1,140
2,934
4,845
7,440
4,920
7,290
7,404
6,750
*
8-Glucuronidase
Activity OD
mS
.029f .001
Not done
960 f 150
10-4
Phenylbutazone
Total =S cprn
Released'
58
45
4
21
12
39
20
3
49
30
8
49
17
0
(tl2)
W9)
10
98
70
43
6
42
8
7
12
.329f .01
.110
.130
.230
.245
.310
.200
.207
.270
.160
.180
.240
.175
.220
.SO
,390
.440
.380
.390
.370
.280
.280
.145
.215
.405
.370
'Additional compounds tested and found to be ineffective at 10-M included: codeine, chlorpheniramine, chlorthiazide, pyribenzarnine or chlorprornazine
TRepresentative SE
SCalculated after the background release (cartilage alone) was subtracted
mately ZOO/, at lO+'M. Pyribenzamine,
chlorothiazide, chlorpromazine, dextropropoxyphene and codeine at 10-SM and
10-4M were all without effect on release of
35Sand p-G.
Gold sodium thiomalate was extremely
effective in suppressing 3% release at
10-4M, 10-5M, and 10-6M but not at
lO-?M (98%, 70%, 48% and 6% inhibi50
tion, respectively (Table 1). There was
however no effect on p-G release at any of
these concentrations. The selective effect of
gold on 35S and not on p-G release is explained by the data given in Table 2 where
it can be seen that gold inhibited directly
the neutral protease activity which was obtained from cell lysates. Hydrocortisone
hemisuccinate inhibited p-G release 55%
Arthritis and Rheumatism, Vol. 17, No. 1 (Januay-Februay 1974)
EFFECT OF ANTIRHEUMATIC DRUGS
Table 2. Effect of Various Compounds
on =S Release from Labeled Ear Cartilage
When Incubated wlth Granule Lysate of
5 x 10' Human Leukocytes
Concentratlon
Drug
(MI
Total
cpm
Percent
inhibition
*
Lysate (leukocytes)'
16,199 958
Lysate (1ymphocytes)t
1,306
Oxyphenbutazone
lo-'
15,246
6
Phenylbutazone
lo-'
14,247
12
lndomethacin
lo-'
14,925
8
Colchicine
lo-'
13,845
14
Aspirin
lod
14,131
19
Hydrocortisone
hemisuccinate
lo-"
18,860 (T13)
Chloroquine SO,
lo-'
11,151
31
Gold Na thiomalate
lo-'
lo-'
1 Qd
Cyclophosphamide
lo-'
15,630
3,126
5,580
14,958
3
80
65
8
neutrophils, 50% lymphocytes, 3% monocytes, 2% eosoniphils from dextran sedimentations
598% lymphocytes -I- 2% monocytes from FicollHypaque fractionation
'45%
and 40y0 at 10-4M and 10-5M, respectivel y (Table 1). Release of 35S was inhibited
only at 10-4M (42%). Hydrocortisone had
a stimulating effect on the cartilage degrading action of the cell-free lysates (Table 2).
Thus although the release of the lysosomal
enzyme marker (8-G)was inhibited, the activity of the residual amount of neutral
protease released was increased. In the intact cell system (Table 1) chloroquine
caused an increase in counts per minute in
the incubation media at lO-4M and 10-5M
concentrations ( 12y0 and 29y0, respectively). The changes in &G release paralleled
the drug's effect on 3% release. Chloroquine
did not have any significant effect at
10-EM.
Granule lysates from 5 x 106 human
leukocytes were incubated with labeled
rabbit ear cartilage for 5 hours (37OC) at
pH 7.4. The lysates of leukocytes induced
a release of 16,200 cpm into the incubation
medium (55% of total), whereas, the lysates
from the same number of mononuclear cells
released only 1306 cpm (Table 2). Oxyphenbutazone, phenylbutazone, indomethacin, colchicine, cyclophosphamide, and aspirin, all at 1 x 10-SM, did not suppress
the 3 5 s release from the labeled cartilage
caused by the human leukocyte granule
lysate (Table 2). Chloroquine was active at
10-3M (.?lo/, inhibition) but not at lO-'M.
Gold sodium thiomalate at a concentration
of 10-4M inhibited the lysate-induced cartilage degradation by 80%. At 10-5M gold
inhibited 35S release by 65%. Hydrocortisone hemisuccinate at 10-4M caused a 13%
increase in soluble counts per minute.
Additional experiments were performed
to determine the effect of a 1 hour preincubation of human leukocytes with various
antiinflammatory agents. Labeled cartilage
was added to the incubation media and the
release of 35Sand P-G determined. Under
these conditions colchicine, phenylbutazone, indomethacin, and aspirin were twoto tenfold more effective in suppressing s5S
and P-G release than when preincubation
was not used. The effect of preincubation
on the action of hydrocortisone was even
more striking since almost a 100-fold increase in activity was observed
M +
10 -6M).
T o determine whether the neutral protease in the supernatant was released from
a preformed pool or was a result of de novo
synthesis, the effect of protein synthesis inhibitors on nonphagocytic enzyme release
was determined. Leukocytes (5 x loa) were
preincubated for 1 hour. Labeled, opsonized cartilage was then added to the reaction vessel. The results given in Table 4
indicate that, as compared to control release (13,122 cpm), puromycin and chloramphenicol at lO-3M and lO-4M had no
Arthrltls and Rheumatism, Vol. 17, No. 1 (January-February 1974)
51
PERPERANDORONSKY
Table 3. Nonphagocytic Enzyme Release by 5 X 10' Human PMN After 1 Hour.
Preincubation with Various Antiinflammatory Agent8
Samples
Concentration
Cartilage alone (no preincubation)
Cartilage 4- preincubated cells
Cartilage non preincubated cells
Cartilage preincubated cells
colchicine
+
-
.036
.360
.360
1OdM
10-M
2,895
7,074
80
35
.110
.230
lO-'M
10-M
4,931
8,250
58
22
.180
.280
1Od
10-M
4,185
7,440
66
30
.170
.270
1O-'M
1o4
6,450
10,212
41
0
,215
.360
1OdM
10%
lO-'M
1OdM
3,807
7,878
6,975
9,435
70
26
35
.160
.270
.245
.310
+
+
+
Cartilage preincubated cells
phenylbutazone Na
+
1,035
10,210
10,410
+
Cartilage preincubated cells
-I- oxyphenbutazone Na
+
Cartilage preincubated cells
f indomethacin Na
+
Cartilage preincubated cells
f hydrocortisone
Cartilage -I- preincubated cells 4- aspirin
9
'After 1 hour preincubation an additional 5-hour incubation was carried out in the presence of IgG
opsonized cartilage as in Table 1.
matoid arthritis, antigenlantibody complexes are found as cytoplasmic inclusions
in infiltrating human leukocytes and in
DISCUSSION
synovial lining cells (8,9).In addition, sodiUnder ordinary circumstances the active um urate crystals are phagocytosed by inmigration of leukocytes to inflammatory filtrating leukocytes in human gout (10).
foci is a protective reaction, since such cells Oronsky, et a1 have demonstrated that huare capable of phagocytosing, degrading, man leukocytes release neutral proteases,
and eliminating potentially pathogenic sub- which are capable of degrading the chonstances, such as microorganisms, crystalline dromucoprotein matrix of cartilage during
material, and antigen/antibody complexes. the process of phagocytosis. Such enzymes
In some cases a potentially protective event are released without loss of cell viability
can cause destructive sequelae. Such may and enzyme release is increased two- to
be the case in inflammatory arthroses where threefold under conditions where the parphagocytosis, or attempted phagocytosis, of ticles to be phagocytosed are affixed to surparticles affixed to the surface of articular faces and cannot be engulfed (nonphagolinings result in the release of enzymes cytic release).
T h e model used in the present expericapable of degrading the matrix of articular
cartilage.
ments was described previously in detail
It has been demonstrated that in rheu- and is one in which human leukocytes ateffect on the release of neutral protease
from human leukocytes.
52
Arthritis and Rheumatism, Vol. 17, No. 1 (January-February 1974)
EFFECT OF ANTIRHEUMATIC DRUGS
Table 4. Effect of Puromycin and
Chloramphenicol on Nonphagocytic
Enzyme Release
Concentration
Drug
Cartilage
Cartilage cells
Puromycin HCI
+
Chloramphenicol Na
succinate
(MI
lod
lo-'
lod
lo-'
Total
cpm
480
13,122
13,283
13,158
15,885
13,683
Percent
inhibition
0
0
(t18)
0
Intact leukocyte suspensions were preincubated
with compounds for 1 hour at 37°C. Incubation
was then continued for an additional 5 hours in
the presence of compound and IgG-coated cartilage.
tempt to phagocytose aggregated human
gamma globulin affixed to the surface of
35S-labeledrabbit ear cartilage (5). T h e release of 35S was shown previously to be a
quantitative and specific marker for the
degradation of cartilage chondromucoprotein (4), whereas the release of p-G was
used as a marker for enzyme release from
lysosomal granules. T h e p-G release is only
a marker system since it is incapable of degrading cartilage matrix, as evidenced by
the fact that during inhibition of neutral
protease by gold the release of p G activity
was unaffected.
In the present experiments various antiinflammatory drugs (steroidal and nonsteroidal) were tested for their capacity to
suppress the release and/or activity of human protease. Phenylbutazone, indomethacin, oxyphenbutazone, and hydrocortisone
at concentrations of 1 0 - 4 ~ all actively
suppressed both neutral protease and @-G
release in a roughly parallel manner. Colchicine was at least tenfold more active
(IO-SM), whereas aspirin was only one
tenth as active (lO-SM). Chloroquine was
totally ineffective and, in fact, it significantl y increased cartilage destruction and p-G
release at a concentration of lO-5M. This
result is somewhat surprising in'light of the
previously reported capacity of chloroquine
to stabilize isolated rat liver lysosomes (1 1).
In addition Chang has shown that rabbit
PMN phagocytosis is inhibited by chloroquine in vitro at concentrations of 10-4M
(12). It should be noted however that human leukocytes have been reported to be
significantly different from rabbit PMNs,
especially with respect to their enzyme constituents (4). Therefore the differences in
chloroquine activity between the present
and previous studies may be a manifestation of species differences.
Cyclophosphamide was ineffective in suppressing cartilage destruction and @-Grelease. This is consistent with the reports
that cyclophosphamide is not effective in
other in vitro systems since only its metabolites have biologic activity (13). Other
classes of drugs, such as codeine, chlorpheniramine, pyribenzamine, and chlorothiazide, were ineffective in preventing cartilage destruction by human leukocytes.
Gold thiomalate at 10-4M prevented the
neutral protease-induced 35S release in the
intact system, however there was no concomitant suppression of p-G release. When
gold was incubated directly with the lysates
from human cells it suppressed s5S release
by 65% at 10-5M. This offers good evidence for a direct effect of gold on neutral
protease activity without interference with
the release process during phagocytosis. All
other compounds thus far tested at lO-SM,
with the possible exception of chloroquine,
did not prevent human cell lysates from
causing chondromucoprotein degradation.
Chloroquine was active at 10-sM (31% inhibition). It is possible that since gold and
chloroquine are actively taken up by human leukocytes and can be demonstrated
in the granule fraction (14,15), the antiinflammatory activity of these drugs might
Arthritis and Rheumatism, Vol. 17, No. 1 (January-February 1874)
53
PERPER AND ORONSKY
be expressed b y their capacity to directly
inhibit the proteolytic enzyme systems intracellularly
It is unlikely that the inhibitory effect of
the various nonsteroidal and steroidal antiinflammatory drugs on release of neutral
protease is due to a direct toxic effect on
the leukocytes since the same compounds
were incapable of inhibiting the cartilagedegrading effect of the cell lysates. Thus
even if the cells had been destroyed by
these agents no inhibition of 85S release
would have been observed. Therefore their
effect had to be at the level of the release
process.
The activity of the various antiinflammatory compounds could be increased twoto tenfold as demonstrated by the increase
in activity of phenylbutazone, indomethacin, oxyphenbutazone, hydrocortisone, and
apirin by an additional 1-hour preincubation of human cells with these compounds
prior to exposure to opsonized cartilage.
Such conditions may be a valid representation of in vivo events since cells circulate
with compound prior to their entry into an
inflammatory site. Under these conditions
phenylbutazone, indomethacin, and oxyphenbutazone demonstrated significant activity at 10-6M, whereas aspirin had significant activity at lO-*M. Peak blood
levels for phenylbutazone after a single
800-mg oral dose is reported to be approximately 125 pg/ml (16) which is greater than
the concentgtion 10-5M that inhibits enzyme release reported in this communication. Aspirin at 10-8M is equivalent to
160pg/ml concentration which can be
achieved with therapeutic doses of salicylates in man (17). In the case of indomethacin 10-6M equals about 3 /*g/ml, again a
concentration that can be achieved with a
single oral therapeutic dose (200 mg) (18).
The concentration of gold used is difficult
to extrapolate to the human system since
.
54
this drug accumulates intracellularly and
in this location its concentration would be
different from that observed in plasma. In
the case of colchicine, which is quite active
as an inhibitor of neutral protease release,
it is impossible to directly extrapolate these
results to the therapy of chronic rheumatoid arthritis since, due to systemic toxicity,
only short term therapy can be employed.
Thus its actual efficacy as an antirheumatic
rather than as an antigout agent cannot be
assessed at this time. It should be noted
however that its antigout efficacy is reported to be related to the prevention of
enzyme release during crystal phagocytosis
(10).
Inhibitors of protein synthesis, puromycin (19), and chloramphenicol (20) did not
prevent cartilage degradation in the model
systems employed. These compounds were
given at concentrations (lO-SM and
1 0 - 4 ~ )that greatly exceeded those necessary to inhibit protein synthesis in vitro.
These data suggest that the neutral protease
in human cells is not synthesized de novo
during phagocytosis but, in fact, is present
in large amounts and stored in the granules
of human leukocytes. It was also shown
that the neutrophil was most likely the cell
type which contained the neutral protease
since intact cells or lysates of a pure population of mononuclear cells were virtually
inactive as a source of neutral protease activity. This is not an unexpected finding
since release only occurs in the presence of
potentially phagocytosable particles (2).
Since the mixed leukocyte preparations
contained approximately 50% lymphocytes, it is apparent that small numbers of
neutrophils are capable of causing significant destruction of cartilage matrix.
This study shows that many commonly
used antirheumatic agents may have, at
least in part, a similar mechanism of action-ie, the suppression of enzyme release
Arthritis and Rheumatism, Vol. 17, No. 1 (January-February 1974)
EFFECT OF ANTIRHEUMATIC DRUGS
from human leukocytes in contact with immobilized immunologic reactants. Certainly
phenylbutazone, oxyphenbutazone, indometha&, colchicine, aspirin, and hydrocortisone would fall into this category. Gold
and to some extent chloroquine may function by directly inhibiting the neutral protease activity of human leukocytes.
REFERENCES
1. Henson PM: Interaction of cells with immune complexes: Adherence, release of constituents and tissue injury. J Exp Med 134:
114+135s, 1971
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effect, matrix, drug, degradation, release, enzymes, leukocytes, cartilage, human, antirheumatic
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