1. Healthcare

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Inhibiting Interleukin-l and Tumor Necrosis Factor-a Does Not Reduce
Induction of Plasminogen Activator Inhibitor Type-l by Endotoxin
in Rats In Vivo
By J.J. Emeis, R. Hoekzema, and A.F. d e Vos
In experimental animals and humans, intravenous (IV) injection of endotoxin induces large increases in circulating plasminogen activator inhibitor type-l (PAI-l), a major inhibitor
of blood fibrinolysis. A similar increase is seen after the injection of interleukin-l (IL-l)or oftumor necrosisfactor-a (TNFa),suggesting that these cytokines mediate the induction,
by endotoxin, of PAI-1.To test this hypothesiswe pretreated
rats, before IV endotoxin, with compounds that inhibit the
formation of cytokines (pentoxifylline; dexamethasone), or
with compounds that inhibit the action of these cytokines
(anti-TNF antiserum for TNF-a; IL-l receptor antagonist for
IL-l). Noneof these pretreatments affected the induction of
PAL1 synthesis by endotoxin. However, pretreatment did
reduce the endotoxin-induced increase in plasma tPA antigen concentration. Thus, the data suggest that, in rats in
vivo, TNF-a and IL-l are not significantly involved in the
induction of PAI-1 by endotoxin.
0 1995 by The American Society of Hematology.
P
nant human IL-lP (lo8 U/mg; a gift from Dr S. Gillis, Immunex,
Seattle, WA) was diluted to 2 pg/mL. Recombinant mouse TNF-a,
rabbit-antimouse TNF-a antiserum, and an enzyme-linked immunosorbent assay (ELISA) kit for mouse TNF-a were obtained from
Genzyme (Boston, MA). Recombinant human IL-l receptor antagonist (IL-lra; a gift from Dr D.E. Tracey, The Upjohn CO,Kalamazoo,
MI) was diluted to 1 mg/mL. Pentoxifylline (Trental) was from
Hoechst AG (Wiesbaden, Germany), and was used as supplied (20
mg/mL). Dexamethasone (Sigma) was first dissolved in ethanol ( I O
mg/mL), and then diluted to 1 mg/mL in saline. Recombinant human
tissue-type plasminogen activator (PA; Activase) was from Genentech(San Francisco, CA). Recombinant rat JMI-229 tPAZ6was a
gift from Dr S.D. Prior (Porton Developments Ltd. Salisbury, UK).
Rabbit-antirat tPA IgG and rat Lz tPA have been described prev i o u ~ l y Rabbit-antirat
.~~
PAI-l IgG was obtained from American
Diagnostica (Greenwich, CT). Biotine, avidin-peroxidase, and tetramethylbenzidine were from Pierce (Rockford, IL).
Animal experimentation. Male Wistar rats (200 to 250 g body
weight) were obtained from the Broekman Institute (Someren, The
Netherlands). All experiments were performed under Nembutal anaesthesia (60 mgkg, intraperitoneally). Injections were administered
into the vein of the penis. Blood was obtained by aortic puncture
into precooled syringes, and anticoagulated with 0.13 m o a trisodium citrate (1 v01to 9 v01of blood). Platelet-poor plasma was
immediately prepared at 4°Cby centrifugation for 10 minutes at
2,00Og, and stored at -20°C. Serum was prepared for 30 minutes
at 37°C and 30 minutes at 4”C, followed by centrifugation for 10
minutes at 2,0008.
Animal experiments had been approved by the Animal Experiments Committee of The Netherlands Organization for Applied Scientific Research TNO, and were in accordance with the guidelines
on animal experimentation presented to the International Committee
of Thrombosis and Haemostasis.”
Experimental design. Rats were injected with a compound under
study, followed-after an interval of 30 to 60 minutes, as detailed
LASMINOGEN ACTIVATOR inhibitor type-1 (PAI-1)
is one of the plasma factors that determine the plasminogen activating potential of blood. Thus, increased blood
levels of this inhibitor are likely to reduce blood fibrinolysis
and thrombolysis, making PAI-1 a potential risk factor for
cardiovascular disease and thromb~embolism.”~
Many types
of insult will induce increased levels of plasma PAI-1 activity, examples of which are sepsis, surgery, and trauma.”6
Gram-negative endotoxemia, both experimentally7.’ andclinical1y?-l6 strongly increases the plasma level of PAI-1 in
humans.
In previous studies, we1’-’’ and other^^^'^-'^ have shown
that the injection of low doses of endotoxin into experimental
animals will induce large and rapid increases of circulating
PAI-1, due to de novo synthesis of PAI-l.24 As a corollary
to these studies we have shown that the endotoxin-induced
cytokines interleukin-la (IL-1a),I7 IL-1P,l9 and tumor necrosis factor-a (TNF-cY)~~
will also increase plasma PA1 activity in rats.
In combination, these observations suggested to us that
the endotoxin-induced increase in PAI-1 activity might be
mediated by endotoxin-induced cytokines, especially IL- 1
and TNF-a. To test this hypothesis we pretreated rats either
with compounds that prevent the synthesis and/or secretion
of cytokines (dexamethasone, pentoxifylline) or with compounds that inhibit the effects of cytokines ( L 1 receptor
antagonist for IL-1; anti-TNF antiserum for TNF-a), and
measured the subsequent endotoxin-induced increase in PAI.
We also measured tissue-type plasminogen activator (tPA)
antigen levels in these rats, because tPA is known to be
increased as well after endotoxin injection. The increase in
PA1 activity was not affected byany of these treatments,
whereas the increase in tPA antigen was. We propose that,
contrary to our hypothesis, the endotoxin-induced cytokines
IL-1 and TNF-a are not significantly involved in the induction of PAI-1 by endotoxin.
MATERIALS AND METHODS
Materials. Endotoxin (lipopolysaccharide [LPS], from Escherichia coli serotype 0128:B12; Sigma, St Louis, MO) was dissolved
in sterile saline to a concentration of 10 pg/mL. All other compounds
were dissolved in sterile saline containing 1% (wdvol) sterile, pyrogen-free human serum albumin (Central Laboratory of the Red Cross
Blood Transfusion Service, Amsterdam, The Netherlands). Recombinant human TNF-a (2.5 X lo7 U/mg protein; a gift from Dr W.
Fiers, Biogent, Gent, Belgium) was diluted to 10 pg/mL. RecombiBlood, Vol 85, No 1 (January l ) , 1995: pp 115-120
From Gaubius Laboratory TNO-PG, Leiden; and Netherlands
Ophthalmic Research Institute, Amsterdam, The Netherlands.
Submitted January 4, 1994; accepted September l , 1994.
Presented in part at the XIthInternational Congress on Fibrinolysis (Copenhagen, Denmark, 1992).
Address reprint requests to J.J. Emeis, PhD, Gaubius Laboratory
TNO-PG, PO Box 430, 2300 AK Leiden, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8501-001I $3.Oo/O
115
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EMEIS, HOEKZEMA, A N D DE VOS
116
Table 1. Effect of Pretreatment With Pentoxifylline or
Dexamethasone on the Induction of TNF-a by Endotoxin
TNF-a (pg/mL)
Pretreatment
Saline
Pentoxifylline
Dexamethasone
LPS
No
controls
Individual Data
Geometric Mean
45,640 28,958
19,281 11,486
770
1,430
992
693
489
339
1,094
307
248 278
<50
< 50
Rats (four per group) were pretreated with pentoxifylline
20,474
(50
mgi
kg IV, 1 hour before LPS), or with
dexamethasone(2 mg/kg intraperitoneally, 2 hours before LPS), and subsequently injected with LPS (10
pg/kg). Serumfor thedetermination of TNF-u concentrations was
obtained 1 hour after LPS injection. For the determination of TNF-a,
see Materials and Methods.
nificantly reduced serumTNF-a levels(measured I hour
after LPS) from a mean value of 20,500 pg/mL in salinepretreated rats to 770 pg/mL (4% of controls) in pentoxifylline-pretreated rats, and to 339 pg/mL (2% of controls) in
1). At 3hoursafter
dexamethasone-pretreatedrats(Table
LPS,mean (n = 3) serum TNF-a valueswere 132 pg/mL
in saline-pretreatedrats and133pg/mL in pentoxifyllinepretreatedrats,whereas
no TNF-a wasdetected in dexamethasone-pretreated animals. Closely similar values were
found when TNF-a levels were determined by ELISA, using
anELISAkit
for mouse TNF-a which also measures rat
TNF-a (data not shown).
Effect oj' LPS on plasma PAI activity. LPS ( 10 ,ug/kg)
induced, as we described p r e ~ i o u s l y , ' ~a ~
large
~ " increase in
plasma PA1 activity. In the present study, the plasma PA1
activity averaged, in all saline-pretreated animals combined,
12 1 U/mL at 3 hours after LPS injection, representing a 25fold increase over the baseline level of 4 to 6 U/mL. The
identification of the increasedinhibitoryactivityas
PAI-I
activity has been described,'"24and couldin the present study
be confirmed by quenching the LPS-inducedactivity by rabbit-antirat PAI-l IgG (not shown).
Effect ofpentoxihlline and dexamethasone on PAI induction b y LPS. Pretreatmentwithpentoxifylline
(20, 50, or
100 mg/kg IV, 1 hour before LPS) had no effectonthe
induction of PA1 by LPS. Saline-pretreated animals had PA1
levels of 134 5 33 U/mL (mean +- SD; n = 9); pentoxifylline-pretreatedratshad
PA1 levels of I18 2 I8 U/mL for
pentoxifyllineat 20 mg/kg(n = 5 ) ; 123 +- 13 U/mLfor
pentoxifylline at 50 mg/kg (n = 4); and 1 1 I 2 21 U/mL for
pentoxifylline at 100 mg/kg (n = 4). These differences were
not significant by one-way ANOVA. Injection of only pentoxifylline didnot affect plasma PA1 activity,as we described previously.3'Acute pretreatment with dexamethasone (2 mg/kg intraperitoneally 2 hours before LPS) had no
effect on the induction of PA1 by LPS (Table 2), nor did
subchronic pretreatment with dexamethasone ( 2 mg/kg intraperitoneally, once daily for 4 days) (Table 2 ) . Corticosterone
had no effect either (Table2). Dexamethasone alone(without
LPS) increased PA1 activity about twofold.24
below-by the intravenous (IV) injection of LPS( I O pgikg), recombinant human TNF-a (10 pgkg), or recombinant human IL-lp (1
pgikg). Bloodfortheassayof
PAI-lactivity was obtained 3
hours'7.'*later. To assess the effect of pretreatment on the induction
of TNF-a by LPS, rats were bled 1 hour after LPS.
TNF-a assay. TNF-a concentrations were determined in serum
by a cytotoxicity assay, usinga subclone of the WEHI 164/13 mouse
fibrosarcoma cell line as the indicator cell, essentially as described
by EspevikandNissen-Meyer."Theassaywascalibratedusing
recombinant mouse TNF-a, and had a lower limit of sensitivity of
l pg/mL. Rat serum samples were assayed at dilutions of l:lO, SO,
250,and1,250. In theserumsamplesTNF-awasspecificallydetected at levels 250 pg/mL.
PA1 assay. PlasmaPA1concentrationsweredetermined
in appropriately diluted plasma by titration with human recombinant tPA
(Activase), followed by determination of the residual tPA activity,
as described by Verheijen et al."' Results will be expressed as units
(U) per milliliter, 1 U/mL being equivalent to 1 ngoftPA inhibited
per mL of plasma.
tPA antigen assay. RattPAantigenwasdetermined
by an
ELISA, as follows. Microtiter plates were coated with rabbit-antirat
tPA IgGZ7(4 pg/mL in carbonate buffer, pH = 9.0) overnightat 4°C
andwashed.Samples(dilutedinphosphate-bufferedsaline[PBS]
containing 5 mg/mL casein) wereincubatedovernightat
4°C and
washed. Bound tPA was subsequently quantitated using biotinylated
Table 2. Effect of Dexamethasone and Corticosteroneon the
rabbit-antirattPA IgG, followedby avidin-peroxidase and tetramethInduction of Plasma PA1 Activity by Endotoxin
ylbenzidine. Rat tPA purified from Lz cells (0.5 to 4.0 ng/mL) was
used as standard. The rat Lz tPA had been calibrated against recombiPA1 Activity (U/mL)
No. of Rats
nant rat JMI-229 tPA.2bResults will be expressed as nanograms of
8 111 t- 28
Ethanol controls
rat tPA per milliliter.
102 2 24
8
Dexamethasone*
Statistics. Statisticalsignificance of differences betweengroups
133 i 11
8
Ethanol
controls
will be analysed by Student's t-test, or by one-wayANOVA fol97 t 24
8
Dexamethasonet
lowed by Bonferroni's modified t-test,as indicated. Differences will
105 -C 11
5
Ethanol controls
be considered significant if P (two-sided) < .OS.
112 t- 16
5
Corticosterone*
RESULTS
Effect of pentoxihlline and dexamethasone on TNF-a induction. A pilot experiment (not shown) demonstrated that
peak TNF-a levels were found 1 hour after the injection of
in
LPS ata dose of 10 pgkg. No TNF-a was detectable
saline-injected controls. Pretreatment of rats with pentoxifylline (50 m g k g IV 1 hour before LPS), or with dexamethasone (2 m g k g intraperitoneally2hours
before LPS), sig-
All data shown are mean 2 SD. Rats were pretreated with corticosteroids as indicated, followed by LPS (10 pg/kg IV). Plasma for PA1
determinations was obtained 3 hours after LPS.For further details,
see Materials and Methods. No significant differences betweentreatment and control groups were present.
* Dexamethasone 2 mg/kg intraperitoneally, daily for 4 days before
LPS (10 pg/kg IV).
t Dexamethasone 2 mg/kg intraperitoneally, 2 hours before LPS.
+ Corticosterone 50 mg/kg intraperitoneally, 2 hours before LPS.
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117
LPS, PAI-1,ANDCYTOKINES
Table 3. Effect of Anti-TNF Antiserum, Interleukin-l-Receptor
Antagonist, or Both, on PA1 Induction
1. Saline, then LPS
2. Control rabbit serum, then
LPS
3a. Anti-TNF antiserum (0.1 mL),
then LPS
3b. Anti-TNF antiserum (0.3 mL),
then LPS
4a. IL-lra (1 mg/kg), then LPS
4b. IL-lra (4 mg/kg), then LPS
5. Anti-TNF (0.1 mL) plus IL-lra
(1 mglkg), then LPS
6. Control rabbit serum, then
TNF
7a. Anti-TNF antiserum (0.1 mL),
then TNF
7b. Anti-TNF antiserum (0.3 mL),
then TNF
8. Saline, then IL-l
9a. IL-lra (1 mg/kg), then IL-l
9b. IL-lra (4 mg/kg), then IL-l
PA1 Activity
WmL)
No. of
Rats
131 2 45
10
1452 5
4
136 2 57
4
113; 141
154 2 32
133; 148
2
4
2
156 2 8
4
52 -c 7
4
30 2 4
4
5; 8
16 -C 2
10 2 1
5; 6
All data shown are mean 2 SD, or individualvalues of two animals.
No significant differences were present between groups 1-5 by oneway ANOVA. Group 6 was significantly different from group 7a. and
group 8 from group 9a. by Student‘s t-test. Rats were pretreated with
anti-TNF antiserum (0.1 or 0.3 murat), with control rabbitserum (0.1
murat), with IL-Ira (1 or 4 mg/kg), or with saline, followed by either
LPS (10 pg/kg), recombinant human TNF-a (10 pg/kg), or recombinant
human IL-1/3 (1 pgikg).Rats were killed 3 hours after LPS, TNF or IL1. For further details, see Materials and Methods.
Effect of anti-TNF antiserum, IL-I receptor antagonist
(IL-Ira), or both, on PAI induction by LPS. Pretreatment
of rats with an antimouse-TNF antiserum (0.1 mL/rat) had
no significant effect on the induction of PA1 by LPS, compared to pretreatment with control rabbit serum (Table 3).
Higher doses of the antiserum (0.15 or 0.3 mUrat, n = 2
per dose) had noeffect either (Table 3). The anti-TNF antiserum did reduce, though, the induction ofPA1 activity by
recombinant human TNF-a, inhibiting partially at a dose of
0.1 mL/rat and fully at a dose of 0.3 mL/rat (Table 3). At a
dose of 1 mgkg, recombinant human IL-lra significantly
inhibited the induction of PAI-l activity by recombinant
human IL-Ip (1 & k g ) , whereas at a dose of 4 mgkg the
receptor antagonist fully inhibited the induction by IL-1p
(Table 3). In contrast, IL-lra did not affect the induction of
PA1by LPS at all (Table 3). Combined pretreatment with
anti-TNF and IL-lra had no effect on the induction of PA1
by LPS either (Table 3).
Additive effects of TNF-a and IL-IP. To see whether
residual circulating levels of one cytokine would potentiate
the other, rats (two per group) were injected with TNF-a
(10 & k g ) with or without simultaneous injection of a small
dose of IL-lp (0.1 pgkg), or with only IL-1p (0.1 Fgkg).
No potentiating effect on PA1 induction was observed: TNFa alone gave PA1 activities of 18 and 20 U/mL; IL-1p alone
8 and 11 U/mL; TNF-a plus IL-lp 19 and 20 U/&. Sirni-
larly, no potentiation of IL-lp (1 & k g ) by a small dose of
TNF-a (1 pgkg) was seen: IL-1p alone gave PA1 activities
of 25 and 25 U/mL; TNF-a alone 12 and 13 U/mL; IL-lp
plus TNF-a 30 and 38 U/mL. IL-lp (1 & k g ) plus TNF-a
(10 & k g ) gave 35 and 42 U/mL. These data suggest that
the combined effect of IL-1p and TNF-a is additive rather
than synergistic. This makes it unlikely that residual amounts
of TNF-a activity [remaining after anti-TNF treatment)
would potentiate IL-lp, or that residual L - l p activity (after
IL-lra treatment) would potentiate TNF-a. The effect of
TNF-a on PAI-1 was linearly related to the injected dose of
TNF-a over the dose-range 0.5 to 60 , & k g (n = 9; r =
.895; P < .01).
Changes in tPA antigen concentrations. LPS induced a
time-dependent increase in tPA antigen (Fig l), preceding
the induction of PA1 which commences only after 1 hour.”
Increases in tPA antigen of a magnitude similar to that induced by LPS were found at 3 hours after the injection of
IL-1p and TNF-a (Table 4). The tPA increase after LPS
was partly inhibited by pentoxifylline, dexamethasone, antiTNF antiserum, and IL-l-ra (Table 4). Similarly, anti-TNF
antiserum and IL-l-ra partly inhibited the tPA increase induced by, respectively, TNF-a and IL-lp (Table 4). Inhibition was not complete, presumably because the high PA1
levels in the circulation caused the formation of more tPAPA1 complexes, which are cleared from the circulation more
slowly than uncomplexed tPA.32
DISCUSSION
This study was designed to test the hypothesis that the
induction of PAI-l by endotoxin is mediated by the cytokines
TNF-a and/or IL-1. This hypothesis originated from two
sets of observations. Firstly, the observation that endotoxin,
a potent inducer of PAI-1, is also a potent inducer in vivo
of IL-1 and TNF-a. Secondly, the observation that both
cytokines will increase PAL1 in vivo. IL-la and p have
been shown to induce PAL1 in rats,”*19whereas TNF-a
g.60
0.00
t
A
I
’
0
/
30
60
/l
I
I
90
120
150
160
time (min)
Fig1. Time course of tPA antigen concentrations in rat plasma
after the IV injection of LPS (10 pglkgl. Data shown are mean 2 SD
of four animals, or single values of two animals (at 30 and 90 minutes). tPAantigenwasdeterminedasdescribedinMaterialsand
Methods.
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118
EMEIS, HOEKZEMA, AND DE VOS
Table 4. Effect of Pretreatment on Endotoxin-Induced
tPA Antigen Levels
tPA Antigen
(ngimL1
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Saline-treated control rats
Saline, then LPS
Normal rabbit serum, then LPS
Pentoxifylline, then LPS
Dexamethasone, then LPS
Anti-TNF antiserum, then LPS
IL-lra, then LPS
Control rabbit serum, then TNF
Anti-TNF antiserum, then TNF
Saline, then IL-l
IL-lra, then IL-l
2.4
8.1
8.4
6.2
6.3
6.3
5.8
5.7
3.2
6.2
3.6
i- 0.2
i- 2.5
i- 3.0
t 1.5
? 1.2
? 1.3
2 1.8
-+ 0.3
i- 0.1
i- 1.0
f 0.3
No. of
Rats
4
10
4
4
4
4
4
4
4
3
3
All data shown are mean 2 SD. Groups 2-7 were not significantly
different by one-way ANOVA. When groups 2 and 3 and groups 4-7
v pretreated)
were combined, the two new groups (non-pretreated
were significantly different by Student‘s t-test(P< .01). Group 8 was
significantly different from group 9, and group 10 from group 11, b y
Student‘s t-test ( P < .05).Rats were pretreated with saline, control
rabbit serum (0.1 murat), pentoxifylline (50 mg/kg IV), dexamethasone (2 mg/kg intraperitoneally), anti-TNF antiserum (0.1 murat), or
IL-Ira (1 mg/kg), followed by either LPS (10 pg/kg), recombinant human TNF-(U (10 pg/kg), or recombinant human IL-ID (1 pg/kg). Rats
were killed 3 hours after LPS,TNF, or IL-l. For further details, see
Materials and Methods.
has been shown to induce PAI-I
in mice,’? rat^,'"^^^'^ and
humans.35-3K
However, the present study provided no support
for our hypothesis: all four procedures meant to interfere
with the production and/or action of cytokines (pretreatment
with pentoxifylline, dexamethasone, anti-TNF antiserum, or
IL-I receptor antagonist) all failed to reduce the induction
of PAL1 by endotoxin.
Pentoxifylline inhibitstheinduction,byendotoxin,
of
TNF-a synthesis by inhibiting the transcription of TNF-a
mRNA.” It is effective in all species tested, including humans:’ ~ h i m p a n z e e , ~ ” ~ ’
and rat.43 In this
study,
it
inhibited the induction of TNF-a by 96%(Table l), but
failed to affect the induction of PAI-I by endotoxin. Dexamethasone, which inhibits thetranslation of TNF-a mRNA?
also inhibited the induction of TNF-a (by 98%, Table l), in
agreement with a study in rats which used a similar dosage
regimen?’ But, like pentoxifylline, dexamethasone had no
effect on PA1 induction (Table 2 ) .
In addition, an antimurine-TNF antiserum that also inhibits rat TNF-a had no effect onPA1 induction, though it fully
inhibited the induction of PA1 by (human) TNF-a (Table3).
All in all,thesedatashowedthat
blocking the synthesis
(pentoxifylline, dexamethasone), or the effect (anti-TNFantiserum) of TNF-a did not affect the induction of increased
PAI- 1 synthesis by endotoxin.
Similar to TNF-a, IL-lP
is induced in rats by
albeit moreslowly,peak levels being obtained only at 5
hours after
endotoxin
ie,
after the
peak
level of
PAL1 has been reached (at 3 to 4 hours”). Dexamethasone
also inhibits, as for TNF-a, the induction of IL-1 activity4’
and of IL-l mRNA,4* via effects on translation and secret i ~ n . ~Pentoxifylline
’
is not known to reduce the induction
of IL-I .”) To block the effects of IL-I, we used an IL- 1
receptor antagonists’.52 that effectively inhibits ILI -induced
effects in variety
a
of species,s3
including
However,
no effect on PAI- 1 induction
like dexamethasone, IL- 1 ra had
by endotoxin in this study, though it inhibited the induction
of PA1 by human IL-lP (Table 3).
Endotoxin not onlyinducesincreasedplasmalevels
of
PAI-I, but also oftPA,induction
of tPApreceding that
of PAI-1,
has
as
been observed both
in
and in
chimpanzees.“ Increased tPA antigen levels have also been
described in humans after treatment with TNF-cx.”-’~ In the
present study similar increases were noted after treatment of
rats with endotoxin (Fig l ) , TNF-a, and IL-I@ (Table 4), an
observation not made in ratsbefore. The increaseintPA
antigen after the injection of
TNF-a was reduced by antiTNF antiserum, and the
increase after IL-I@by IL- Ira (Table
4). The increase in tPAantigenafterendotoxin
injection
was reduced, though to a lesser extent, by pretreatment with
pentoxifylline, dexamethasone, anti-TNF antiserum, and ILIra. This suggeststhat pretreatment with anti-TNF antiserum
and IL-Ira was not only effective against injected (human)
TNF-a or IL-l@, but also against endogenous TNF-a and
IL-I . That tPA was not reduced to normal control levels is
presumably due to the fact, mentioned above,that the induction of PAI-1 still occurred in all animals, resulting in increasedcirculatinglevels
of tPA-PA1 complexes thatare
detected by our tPA ELISAassay. Also, it is likelythat
tPA release is induced” after endotoxin injection
by other
endotoxin-induced compounds suchasplatelet-activating
factor, catecholamines, vasopressin, etc.
Because synergistic effects between TNF-a and IL-1 have
been reported (eg, ref 56), and because in our experiments
low residuallevels of cytokines activity are likely to be
present during treatment with antiserum or withreceptor
antagonist, we investigatedwhethersynergistic
effects on
PA1 synthesis couldbe detected. Asdescribedabove, no
such synergy between TNF-a and IL-10 was found, using
various combinations of the two cytokines, but rather additive effects. Moreover, because the PA1 response to TNF-a
was linear over the TNF concentration range 0.5 to 60 ,ug/
kg, we considerit unlikely that residual amounts of TNF (or
IL-I) would significantly have affected the PA1 concentrations.
In a previous publicationz4 we have shown that a variety
of autacoids that are induced or released by endotoxin in vivo
(eg,cyclo-oxygenaseandlipoxygenase
products,plateletactivating factor, catecholamines, histamine, cyclic nucleotides, opioids, vasopressin, thrombin) are not involved in the
induction of PAI-I by endotoxin. It has also been shown
that IL-6 is unable to induce PAL1 in rats in v~vo,’~,’’
as is
the case in vitro.25,’XOur present observations suggest that
IL-1 andTNF-ado
not mediatetheeffect
ofendotoxin
on PAI-l either. Thus, the mechanism by which endotoxin
induces increased synthesis of PAL1 in vivo remains unexplained. This situationisthemoreunfortunatebecauseit
was recently suggested2’ that TNF-a mediates, in chimpanzees, the endotoxin-induced increase in tPA that precedes
the increase in PAL1 activity, an observation in agreement
with our data. It follows that inhibiting TNF-a will, at least
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LPS,PAI-1, AND CMOKINES
inrats,inhibittheprofibrinolytic,tPA-mediated,
effect of
endotoxin without interfering with its antifibrinolytic, PAI1 -mediated effect. In combination, theseeffects might then
shift thefibrinolyticbalanceinblood
even furthertoan
inhibitory state, worsening the procoagulant
effects of endotoxin. It would be of interest to study whetherthis situation,
as described here for rats, also applies to primates and humans.
ACKNOWLEDGMENT
The authors are indebted to Dr W. Fiers (Biogent, Gent, Belgium)
for a gift of recombinant human TNF-a;to Dr S. Gillis (Immunex,
Seattle, WA) for a gift of recombinant human IL-lP; to Dr D.E.
Tracey (The Upjohn CO,Kalamazoo, MI) for providing recombinant
human IL-l receptor antagonist; and to Dr M. Schanharting (Hoechst
AG, Wiesbaden, Germany) for providing pentoxifylline. We thank
M. Bekkers and C.M. van den Hoogen for expert technical assistance.
REFERENCES
1. Wiman B, Hamsten A: Impaired fibrinolysis and risk of thromboembolism. Prog Cardiovasc Dis 34:179, 1991
2. Prins MH, Hirsh J: A critical review of the relationship between
impaired fibrinolysis and myocardial infarction. Am Heart J 122:545,
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1995 85: 115-120
Inhibiting interleukin-1 and tumor necrosis factor-alpha does not
reduce induction of plasminogen activator inhibitor type-1 by
endotoxin in rats in vivo
JJ Emeis, R Hoekzema and AF de Vos
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