Immunology
1993
78
286-290
Malaria
antigen
and
cytokine-induced
production
of
reactive
nitrogen
intermediates
by
murine
macrophages:
no
relevance
to
the
development
of
experimental
cerebral
malaria
P.
G.
KREMSNER,
A.
NUSSLER,*
S.
NEIFER,
M.
F.
CHAVES,
U.
BIENZLE,
G.
SENALDIt
&
G.
E.
GRAUt
Landesinstitutfiur
Tropenmedizin
Berlin,
Germany,
*Department
of
Surgery,
University
of
Pittsburgh,
Pennsylvania,
U.S.A.
t
WHO-Immunology
Research
and
Training
Centre,
Department
of
Pathology,
University
of
Geneva,
Switzerland
Acceptedfor
publication
6
September
1992
SUMMARY
The
in
vitro
production
of
reactive
nitrogen
intermediates
(RNI)
by
murine
macrophages
was
evaluated
in
response
to
heat-stable
malaria
antigen
and
cytokines.
Malaria
antigen,
interferon-y
(IFN-y)
and
tumour
necrosis
factor
(TNF)
induced
RNI
production
in
macrophages
in
a
dose-
dependent
way.
RNI
production
steadily
increased
over
a
2-day
period
and
was
enhanced
when
the
malaria
antigen
was
co-incubated
with
IFN-y
and/or
TNF.
RNI
production
induced
by
either
IFN-y
or
malaria
antigen
or
a
combination
of
the
two
was
suppressed
by
pentoxifylline
in
a
dose-dependent
manner.
Pentoxifylline
did
not
significantly
influence
TNF-induced
RNI
production.
L-N-mono-
methyl
arginine
reduced
malaria
antigen,
IFN-y
and
TNF-induced
RNI
production
when
these
reagents
were
used
in
combination
or
alone.
An
anti-TNF
monoclonal
antibody
(mAb)
reduced
IFN-y-induced
RNI
production,
but
did
not
significantly
alter
the
malaria
antigen-induced
RNI
synthesis
by
macrophages.
The
influence
of
inhibitors
of
nitric
oxide
synthase,
L-N-monomethyl
arginine
and
N
w-nitro-L-arginine,
was
studied
in
experimental
cerebral
malaria.
They
did
not
exert
any
significant
effect
on
the
development
of
cerebral
malaria
in
Plasmodium
berghei
ANKA-infected
CBA/J
mice.
INTRODUCTION
Reactive
nitrogen
intermediates
(RNI)
are
nitric
oxide,
nitrite
and
nitrate,
derived
from
L-arginine
along
a
metabolic
pathway
including
nitric
oxide
synthase
as
the
key
enzyme.
They
can
be
produced
by
many
mammalian
cells.
RNI
are
bioactive
mole-
cules
which
exhibit
in
vitro
cytotoxicity
against
protozoal
parasites
such
as
Toxoplasma
gondii'
and
Leishmania
major.2,3
The
toxic
effects
of
RNI
are
probably
due
to
their
interactions
with
iron-dependent
enzymes
such
as
aconitase
in
the
citric
acid
cycle
or
enzymes
in
the
mitochondrial
respiratory
chain
contain-
ing
iron-sulphur
prosthetic
groups.4
Recently
it
was
demonstrated
that
nitrite
and
nitrate
ions
are
toxic
in
vitro
to
asexual
blood
stages
of
Plasmodiumfalciparum.5
Nissler
et
al.6
showed
RNI-dependent
destruction
of
intrahepa-
tic
P.
yoelii
rodent
parasites
which
was
incited
by
tumour
necrosis
factor
(TNF)
and
interleukin-6
(IL-6).
In
a
similar
study
Mellouk
et
al.7
demonstrated
an
anti-parasitic
effect
of
interferon-y
(IFN-y)
upon
pre-erythrocytic
stages
of
P.
berghei,
which
could
be
neutralized
by
L-N-monomethyl
arginine
(L-
NMMA),
an
inhibitor
of
nitric
oxide
synthase.
Interestingly,
Correspondence:
Dr
P.
G.
Kremsner,
Landesinstitut
fur
Tropenme-
dizin
Berlin,
Engeldamm
62,
1020
Berlin,
Germany.
Clark
et
al.8
hypothesized
intriguing
links
between
RNI
produc-
tion
and
the
development
of
cerebral
malaria.
In
the
present
study
the
in
vitro
effects
of
malaria
antigen
and
cytokines
were
examined
upon
RNI
production
by
macro-
phages,
as
well
as
the
effects
of
anti-TNF
monoclonal
antibody
(mAb)
and
pentoxifylline
both
known
to
inhibit
the
develop-
ment
of
murine
cerebral
malaria.9"0
The
effects
of
L-NMMA
were
also
studied
in
vitro
and,
additionally,
in
vivo
in
the
development
of
experimental
cerebral
malaria.
MATERIALS
AND
METHODS
Malaria
antigen
and
other
modulators
of
RNI
production
Malaria
antigen
was
prepared
as
follows:
P.
vinckei-infected
mice
with
60%
parasitized
erythrocytes
were
bled,
erythrocytes
were
washed
in
sterile
phosphate-buffered
saline
(PBS),
sus-
pended
to
a
concentration
of
101
parasitized
cells/ml,
and
incubated
in
PBS
for
24
hr
at
370
in
5%
CO2.
Supernatant
was
then
removed,
centrifuged,
boiled
for
5
min,
passed
through
a
0-2
iim
pore
filter
(Millipore,
Eschborn,
Germany),
and
stored
at
4'
before
use.'0
To
eliminate
any
lipopolysaccharide
(LPS)
contamination
in
the
malaria
antigen
preparation,
5
,ug/ml
polymyxin
B
(Sigma,
Deisenhofen,
Germany)
was
added
to
all
286
Reactive
nitrogen
intermediates
and
experimental
cerebral
malaria
culture
media."
LPS
(Sigma)
was
used
to
check
the
neutralizing
capacity
of
polymyxin
B.
In
addition
malaria-immune
serum
was
generated
by
the
i.p.
injection
of
200
pl
malaria
antigen
9
days
before
bleeding
of
mice.
The
inhibitory
capacity
of
this
immune
serum
was
investigated
on
malaria
antigen
and
LPS-
induced
RNI
production
by
macrophages
in
vitro.
Recombinant
murine
TNF
and
IFN-y
(Dr
G.
R.
Adolf,
Boehringer-Ingel-
heim,
Vienna,
Austria)
were
tested
as
stimulants
in
the
concen-
trations
given
below.
A
rat
anti-mouse
TNF mAb
(Vlq,
Dr
P.
H.
Krammer,
DKFZ
Heidelberg,
Germany)
was
used
in
a
dilution
that
neutralized
2000
U/ml
TNF.
Pentoxifylline
(Rentschler,
Laupheim,
Germany)
and
L-NMMA
(Dr
S.
Moncada
and
Dr.
F.
Y.
Liew,
Wellcome
Research
Laborator-
ies,
Beckenham,
U.K.)
were
tested
as
inhibitors
of
RNI
production
in
a
dose
range
as
indicated
in
Results.
Modulation
of
RN!
production
by
macrophages
Ten-week-old
female
BALB/c
mice
(Bundesgesundheitsamt
Berlin,
Germany)
and
CBA/J
mice
(Shaw's
Farm,
Blackthorn,
U.K.)
were
injected
with
3
ml
2%
amylose
intraperitoneally
3-5
days
prior
to
macrophage
harvesting.
Peritoneal
cells
were
removed
in
S
ml
cold
RPMI-1640
(Gibco,
Gaithersburg,
MD)
containing
polymyxin
B,
washed
and
resuspended
in
CRPMI
(RPMI-1640
with
10%
heat-inactivated
foetal
calf
serum)
to
2
5
x
106
cells/ml.
The
cell
suspension
was
aliquoted
in
100-Pl
portions
onto
a
96-well
plate
(Nunc,
Wiesbaden,
Germany)
and
allowed
to
adhere
at
37°
in
the
presence
of
5%
CO2
for
approximately
3
hr,
after
which
cells
were
washed
twice
with
medium.
Remaining
adherent
macrophages
covered
approxi-
mately
one-third
of
the
well
surface
area.
Macrophage
cultures
were
stimulated
with
100
pl
aliquots
containing
malaria
antigen
and
other
stimulants
in
concentra-
tions
as
indicated
in
Results.
Experiments
were
performed
three
to
five
times
in
quadruplicates.
After
different
incubation
periods,
supernatants
were
removed
to
measure
RNI.
Nitrite
determination
Cell-free
culture
media
were
assayed
for
nitrite,
the
stable
end
product
of
the
nitrogen
oxidation
of
L-arginine,
with
use
of
the
Griess
reagent.
Briefly,
100
,l
aliquots
were
added
to
200
,l
of
a
1:
1
mixture
combined
immediately
beforehand
of
1%
sulphani-
lamide
and
01%
N-1-naphthylethylenediamine
in
45%
acetic
acid.
Absorbance
was
read
immediately
at
540
nm,
cross
filtered
at
690
nm.
A
concentration
gradient
for
NaNO2
in
medium
demonstrated
a
linear
relationship
to
absorbance,
and
was
used
as
a
standard
curve.
Statistical
analysis
Statistical
analysis
was
performed
using
the
Wilcoxon
rank
sum
test
and
the
Fisher's
exact
test.
Treatment
of
experimental
cerebral
malaria
by
L-NMMA
Eight-week-old
female
CBA/J
mice
(Bomholtgard,
Ry,
Den-
mark)
were
infected
by
i.p.
inoculation
of
106
P.
berghei
ANKA
infected
erythrocytes.
Approximately
80%
of
mice
develop
acute
cerebral
malaria
within
the
second
week
with
a
parasitae-
mia
of
only
5-10%.12
Infected
mice
were
injected
i.p.
on
day
6
with
S
mg
L-NMMA
(n=
10)
or
5
mg
N
w-nitro-L-arginine
(NNA;
Sigma,
Buchs,
Switzerland;
n
=
14)
dissolved
in
normal
saline.
The
dose
of
5
mg
was
chosen
because
it
demonstrated
in
vivo
effects
in
a
LPS
toxicity
model.
Control
mice
received
normal
saline
(n
=
14).
In
a
second
experiment
infected
mice
received
two
i.p.
injections
daily
starting
on
day
1
until
day
9 of
either
L-NMMA
(2
mg
solved
in
200
,l
normal
saline;
n
=
10)
or
D-NMMA
(Drs
Moncada
and
Liew,
Wellcome;
2
mg
in
normal
saline;
n
=
10)
or
L-arginine
(Drs
Moncada
and
Liew,
Wellcome;
2
mg
in
normal
saline;
n=
10).
D-NMMA
does
not
inhibit
the
L-arginine-
dependent
production
of
RNI.3
RESULTS
RNI
production
by
macrophages
stimulated
with
malaria
antigen
and
cytokines
Spontaneous
RNI
production
by
peritoneal
macrophages
cul-
tured
in
CRPMI
medium
served
as
a
control.
In
several
experiments
(n
=
5)
malaria
antigen
induced
the
RNI
synthesis
to
detectable
levels
after
only
1
day.
Optimal
induction
of
RNI
synthesis
was
achieved
after
2
days,
therefore
data
are
given
for
this
time-point
unless
otherwise
indicated.
Polymyxin
B
only
slightly
reduced
the
capacity
of
malaria
antigen
to
induce
RNI
production
(25%),
whereas
the
effect
of
01
,ug/ml
LPS
was
completely
abolished
by
5
pg/ml
polymyxin
B.
RNI
production
was
decreased
by
50%
after
30
min
preincubation
of
malaria
antigen
with
immune
serum,
diluted
1:50.
However,
the
LPS-
induced
RNI
production
by
macrophages
was
not
reduced
using
the
same
serum
(data
not
shown).
Malaria
antigen
induced
RNI
synthesis
by
macrophages
of
BALB/c
mice
in
a
dose-dependent
manner
(Fig.
1).
Both
cytokines,
IFN-y
and
TNF
also
induced
dose-dependent
RNI
production.
RNI
were
significantly
increased
in
an
additive
way
by
co-incubation
of
cytokines
with
malaria
antigen
(P
<005,
Fig.
1).
The
highest
level
of
RNI
was
observed
after
stimulation
with
malaria
antigen
plus
100
U
IFN-y/ml
(61-2+0
4
ym
NO2-).
These
findings
were
confirmed
by
identical
experiments
in
BALB/c
and
CBA/J
mice
without
differences
between
mouse
strains.
70
-
60
-
50
-
2
40-
0
30-
z
20
-
1
0
-
nfl
Ri
I
medium
malaria
antigen
IFN-y
TNF
t:25
1:50
1:100
100
10
1
1000
100
units/ml
Figure
1.
Nitrite
(NO2-)
production
by
peritoneal
macrophages
of
BALB/c
mice
cultured
for
48
hr
with
different
agents:
CRPMI
medium
with
polymyxin
B
served
as
a
reference
for
the
spontaneous
NO2-
production.
Malaria
antigen,
IFN-y
and
TNF
were
either
used
alone
(0)
or
in
combination
(1a).
The
values
shown
are
means
with
the
upper
range
of
quadruplicates
of
a
representative
experiment.
I
287
P.
G.
Kremsner
et
al.
0
z
10
n
10-
medium
malaria
antigen
IFN-y
alone
anti-TNF
PXF
alone
anti-TNF
PXF
Figure
2.
Nitrite
(NO2-)
production
by
peritoneal
macrophages
of
BALB/c
mice
cultured
for
24
hr
in
the
presence
of
malaria
antigen
(1:
20)
and
anti-TNF
or
pentoxifylline
(1
mg/ml)
and
in
the
presence
of
IFN-y
(100
U/ml)
and
anti-TNF
or
pentoxifylline
(1
mg/ml).
The
values
shown
are
means
with
the
upper
range
of
a
representative
experiment.
PXF,
pentoxifylline.
70-
60
-
50-
:
40
0
30
z
20-
10-
100
10
units
IFN-y/ml
1
0I1
Figure
3.
Nitrite
(NO2-)
production
by
peritoneal
macrophages
of
BALB/c
mice
cultured
for
48
hr
in
the
presence
of
malaria
antigen
and
different
doses
of
IFN-y
with
and
without
pentoxifylline:
IFN-y
+ma-
laria
antigen
(0);
IFN-y
+
malaria
antigen
+
pentoxifylline
(0-
mg/ml)
(A);
IFN-y+malaria
antigen+pentoxifylline
(1
mg/ml)
(O)
.
The
values
depicted
are
means
of
quadruplicates
of
a
representative
experiment.
Table
1.
The
influence
of
L-NMMA,
D-NMMA
and
L-arginine
on
the
development
of
experimental
cere-
bral
malaria
(n
=
1O/group).
The
drugs
were
injected
i.p.
2
mg
twice
a
day
starting
on
day
until
day
9.
The
numbers
indicate
surviving
mice/group
Days
after
infection
Treatment
5
6
7
8
9
10
11
RNI
production
by
macrophages
inhibited
by
anti-TNF
mAb,
pentoxifylline
and
L-NMMA
Experiments
were
performed
with
macrophages
of
BALB/c
mice.
Anti-TNF
mAb
alone
did
not
affect
the
spontaneous
release
of
RNI
by
macrophages,
but
it
significantly
inhibited
the
IFN-y
induced
RNI
production
(P
<
0
05,
Fig.
2).
Pentoxifylline
(1
mg/ml)
significantly
reduced
both
IFN-y
and
malaria
antigen-induced
RNI
production
of
macrophages
(P
<
0
05,
Fig.
2),
but
it
did
not
reduce
the
TNF-induced
RNI
synthesis
(data
not
shown).
In
further
experiments
(n
=
5),
concentrations
of
up
to
1
mg/ml
pentoxifylline
showed
no
inhibitory
effect
on
spontaneous
RNI
production
of
macro-
phages.
However,
pentoxifylline
exhibited
a
dose-dependent
significant
suppression
of
RNI
production
of
macrophages
stimulated
with
malaria
antigen
(1:
100)
plus
various
concentra-
tions
of
IFN-y
(Fig.
3).
In
macrophage
cultures
using
IFN-y
(10
U/ml)
and
malaria
antigen
(1:100)
for
stimulation
(100%
value)
a
concentration
gradient
of
L-NMMA
(500-2
uM)
was
used
for
the
inhibition
of
RNI
production.
A
reduction
of
90%
and
80%
was
obtained
at
500
and
100
yM
L-NMMA,
an
intermediate
inhibition
of
40%
at
20
gM,
and
no
inhibition
at
2
pm.
Likewise
L-NMMA
reduced
IFN-y,
TNF
and
malaria
antigen-induced
RNI
production
when
these
reagents
were
used
alone
(data
not
shown).
In
macrophages
cultured
for
or
2
days
without
stimulants
a
reduction
of
spontaneous
RNI
production
to
background
levels
of
medium
was
found
with
500
yM
L-NMMA.
The
effect
of
the
treatment
of
experimental
cerebral
malaria
by
L-
NMMA
Single
injections
of
either
5
mg
L-NMMA
or
5
mg
NNA
on
day
6
after
infection
did
not
influence
the
development
of
murine
cerebral
malaria.
Seventy-six
per
cent
of
all
mice
died
with
the
syndrome
of
cerebral
malaria
between
days
7
and
9.
There
was
no
difference
between
treated
mice
and
control
mice
(data
not
shown).
Moreover,
using
a
different
protocol
of
L-NMMA
administration
(2
mg
twice
daily
from
day
until
death)
mice
were
no
more
protected
against
death
from
cerebral
malaria
than
animals
receiving
D-NMMA
or
L-arginine
or
normal
saline,
respectively
(Table
1).
On
the
contrary,
although
not
reaching
the
level
of
statistical
significance
(P
>
0-05),
L-
NMMA-treated
mice
died
of
cerebral
malaria
earlier.
DISCUSSION
In
the
present
study
an
additive
stimulatory
effect
of
heat-stable
malaria
antigen
and
cytokines
on
RNI
production
of
murine
macrophages
was
demonstrated.
This
parallels
findings
in
one
study
in
which
macrophages
were
stimulated
with
LPS
and
IFN-y4
and
in
another,
in
which
Leishmania
major
amastigotes
and
IFN-y
were
used
as
stimulants.3
However,
in
these
studies
a
potentiating
synergistic
effect
of
IFN-y
and
LPS
or
amastigotes
on
RNI
production
was
shown.
Results
of
our
in
vitro
investigations
support
evidence
for
the
hypothesis
of
Mellouk
et
al.7
that
sequestered
malaria
parasites,
like
Leishmania,
might
provide
their
own
second
activation
signal
for
maximum
RNI
production
by
various
cells.
The
malaria
antigen
used
in
the
present
study
is
a
heat-stable
antigen
mixture
of
culture
supernatant
from
P.
vinckei.
This
antigen
preparation
induced
macrophages
to
secrete
comparable
amounts
of
TNF
as
did
a
L-NMMA
10
9 6
0
0
0
0
D-NMMA
10
7
3
1
1
1
0
L-arginine
10
9
6
2
2
1
0
None
10
10
5
4
1
1 1
288
Reactive
nitrogen
intermediates
and
experimental
cerebral
malaria
289
similar
antigen
preparation
from
P.
berghei
(P.
G.
Kremsner,
unpublished
results).
Moreover,
the
components
of
exoantigen-
activating
macrophages
are
phospholipids.
They
seem
to
be
closely
related
between
different
species
of
Plasmodium.'3
In
contrast
to
the
L-arginine
analogue
L-NMMA,
anti-TNF
mAb
and
the
phosphodiesterase
inhibitor
pentoxifylline
did
not
reduce
the
baseline
RNI
production
by
macrophages.
It
is
therefore
assumed
that
the
latter
agents
exert
an
inhibitory
influence
on
stimulation
of
RNI
production
rather
than
on
spontaneous
production.
However,
differences
exist
between
pentoxifylline
and
anti-TNF
as
to
the
position
where
they
exert
their
inhibitory
action
on
RNI
production
of
macrophages.
Pentoxifylline
reduced
malaria
antigen
and
IFN-y
induced
RNI
production,
but
not
TNF-induced
RNI
production.
In
contrast
anti-TNF
mAb
significantly
reduced
IFN-y-induced
but
not
malaria
antigen-induced
RNI
production
of
macrophages.
Evidence
is
accumulating
that
RNI
are
major
antiplasmo-
dial
effector
molecules
in
asexual
blood5
and
hepatic
stages
of
malaria.6
7
Thus,
it
is
conceivable
that
part
of
the
anti-parasitic
effect
of
IFN-y
and
TNF
observed
in
different
murine
malaria
infections'4-'6
may
well
be
mediated
by
RNI.
However,
IFN-y
and
TNF
are
also
involved
in
the
development
of
cerebral
malaria.
This
became
obvious
when
neutralizing
antibodies
to
either
IFN-y
or
TNF
were
found
to
prevent
cerebral
malaria
in
P.
berghei
ANKA-infected
CBA/Ca
mice.9"7
In
addition
malaria-immune
serum
as
well
as
pentoxifylline
also
prevented
murine
cerebral
malaria.'0"8
All
these
regimens
substantially
decrease
RNI
production
possibly
via
different
pathways,
as
may
be
inferred
directly
or
indirectly
from
our
in
vitro
studies.
Clark
et
al.8
hypothesized
that
cerebral
malaria
may
be
induced
by
RNI
originating
from
endothelial
cells
lining
cerebral
vessels
and
from
mononuclear
cells
concentrating
in
small
cerebral
vessels
during
malaria.
In
view
of
our
results,
which
show
no
significant
influence
of
substances
like
L-NMMA
and
NNA,
which
specifically
inhibit
RNI
synthesis,
on
the
course
of
murine
cerebral
malaria,
it
is
assumed
that
RNI
do
not
play
a
major
causative
role
in
this
syndrome.
This
is
supported
by
the
observation
that
inhibitors
of
RNI
production,
used
in
doses
similar
to
those
in
the
present
study,
exerted
biological
effects
in
a
LPS
mouse
toxicity
model
with
a
single
injection
regimen
(A.
Niissler,
unpublished
results)
and
in
murine
P.
vinckei
malaria
daily
injections
of
L-NMMA
led
to
an
aggravation
of
malaria
organ
pathology
and
death
despite
effective
chemotherapy.'9
Enhanced
organ
pathology
after
L-NMMA
injection
has
also
been
reported
in
mice
previously
injected
with
LPS.20
In
rats
with
endotoxic
shock
similar
doses
of
L-NMMA
accelerated
the
decline
in
blood
pressure
with
all
animals
dying
earlier
than
controls.2'
Our
concept
that
RNI
protect
against
malaria-
associated
complications
such
as
cerebral
malaria
rather
than
induce
it,
is
also
supported
by
other
data.
The
inhibition
of
RNI
production
in
vivo
increased
leucocyte
adherence
to
endothe-
hum
via
CD11/CD18
integrins
more
than
15-fold.22
Experi-
mental
cerebral
malaria
was
abrogated
by
antibodies
to
CD
I
I
a,
the
a-chain
of
the
integrin
leucocyte
function-antigen
1,
indicat-
ing
that
CD1
la/CD18-mediated
adhesion
of
leucocytes
to
endothelium
is
critical
in
the
pathogenesis
of
cerebral
malaria.23'24
IL-
1
can
induce
RNI
production.25
Murine
cerebral
malaria
was
prevented
by
a
low-dose
therapy
of
IL-1,26
which
could
have
been
mediated
by
the
stimulation
of
RNI
produc-
tion.
RNI
were
first
described
as
endothelium
derived
relaxing
factor
leading
to
vasodilation
of
blood
vessels.27
Pathological
spastic
constriction
of
intracerebral
arterioles
was
identified
as
being
a
major
cause
leading
to
clinical
(P.
falciparum)
and
experimental
(P.
berghei)
cerebral
malaria.28
Prostacyclin
and
RNI
have
similar
functions
such
as
the
induction
of
endothe-
lium-dependent
relaxation
and
the
inhibition
of
platelet
aggre-
gation.29
Experimental
cerebral
malaria
was
prevented
by
administration
of
a
stable
prostacyclin
analogue.30
Taken
together,
these
data
suggest
that
RNI
do
not
play
a
critical
role
in
the
pathogenesis
of
neurovascular
lesions
charac-
teristic
of
murine
cerebral
malaria.
However,
in
this
experi-
mental
model,
only
end-stage
vascular
problems
can
be
assessed:
early
stages
of
neurological
disturbances
go
unnoticed.
One
cannot
exclude,
therefore,
that
RNI
might
explain
meta-
bolic
changes
taking
place
at
very
early
stages
of
brain
complications,
i.e.
those
observed
in
patients
with
cerebral
malaria
who
fully
recover
from
their
acute
episode.
Studies
of
RNI
production
in
these
patients
are
required
to
clarify
this
point.
ACKNOWLEDGMENTS
We
thank
Dr
F.
Rosenkaimer
(Boehringer
Ingelheim,
Germany)
and
Dr
W.
Herrmann
(Rentschler,
Laupheim,
Germany)
for
support
and
T.
Rasenack
and
S.
Bantz
for
technical
assistance.
We
are
grateful
to
Dr
G.
R.
Adolf
(Boehringer,
Vienna,
Austria)
for
the
recombinant
cytokines;
to
Dr
P.
H.
Krammer
(DKFZ
Heidelberg,
Germany)
for
the
anti-TNF
mAb;
and
Dr
S.
Moncada
and
Dr
F.
Y.
Liew
(Wellcome,
Beckenham,
U.K.)
for
provision
of
L-NMMA,
D-NMMA
and
L-
arginine.
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