Daniel G. Remick and Lorelie Villarete .... disease;. L-NAME,. (L-N-nitroarginine methyl ester. Reprint requests: Daniel. Remick,. M.D., .... the Fenton reaction.
Regulation of cytokine gene expression and reactive nitrogen intermediates Daniel
G. Remick
Department
Abstract:
and Lorelie
of Pathology,
Reactive
oxygen
University
of Michigan
intermediates
Medical
(RO!),
re-
(RNI), and cytokines at sites of acute inflamma-
tion. Previous between the
of
sequent recent
generation data indicates
as end-stage tion
and
tissue
inhibit
molecules
injury,
but
type
(DMSO) stimulated
!!
increase tion
of !L-8, erating
and
of
and
RN!
in these
synthase
will
same
cells.
decrease
[5, 6]. Interestingly,
that
the
not
Reactive intermediates
IL-8
recruiter
of
at
duce
substantial
gether
with
Inhibi-
(NO)-genof IL-8.
.
hydroxyl
The
and activation
recruitment
inflammation cytokines
of neutrophils
remains have been as tumor
(PMNs)
to sites
of active
defined with
process. neutrophil
Certain recruit-
a poorly associated
ment,
such
(IL-i) attract
[1], but careful experimentation has shown that they neutrophils through the induction of another newly
necrosis
factor
(TNF)
or interleukin-l
translated protein mediator [2]. Interleukin-8 (IL-8) represents a newly described cytokine [3] and is a potent inducer of neutrophil chemotaxis. It is produced early in inflammation such stimuli up-regulated
as a direct result of interaction as endotoxin [4], and it will by the early, alarm cytokines
of cells with also be further such as TNF
of ROIs
proteases
[ii,
[13],
12],
in
which,
injure
the
RO!s of both activation
gene
factors including oxyR [14]. The predicted
lows:
low
serve duces
to up-regulate expression neutrophil chemotaxis.
intracellular
to-
tissue.
damage role Our
nitric
oxide;
factor; chronic
previous
work
it merely
reactive IL,
PMN,
LPS,
oxygen
interleukin;
and
RNI
the
ROI/RNIs
expands
and
the
RNI,
dimethyl
neutrophils; L-NAME,
in-
generation
intermediates;
SOD,
their
cascade that elicits an intimate link be-
DMSO,
polymorphonuclear disease;
ROl
concerning
RNI,
lipopolysaccharide;
granulomalous
of
[15-17],
as initiators of data demonstrates
ROl,
intermediates;
in fact
prokaryotic of various
of IL-8, which then inThis hypothesis does not in
production of ROl, chemoattractants.
Abbreviations: trogen
have
and nuclear factorsequence is as fol-
concentrations
contradict
tween the neutrophil
of neutrophils
nitrogen frequent
(reviewed
expression.
flammatory neutrophils.
Recruitment
inflammation
quantities
cytokine
tissue
superoxide
of acute
reactive are also
shown to be important inducers eukaryotic gene expression by
way
.
sites
days
of inflammation. (ROl), injury
regulating
and
interleukin-6
to sites
for several
there is a theoretia role for IL-8 as
been and
any
.
to persist
tors
Key
ar,4ioxidants
of lipopolysac-
However, ROI/RNI may serve a completely different function in acute inflammation, where they do not operate solely as end-stage effector molecules, but also as media-
transcription KB, respectively
inierleukin-8
the ability
released
will inhibit the NO-driven production of!L-8. data indicate that HO! and RN! can serve as intracellular second messengers to induce IL-8 gene expression. J. Leukoc. Biol. 59: 471-475; 1996.
Our
.
dose
refs. 9 and 10). The accepted hypothesis that links these observations may be stated as follows: PMNs are recruited to sites of acute inflammation; once at the site, they pro-
not
radical appears to be the final common cell activation for IL-8 synthesis, since
Words:
has
of neutrophils
will
DMSO
radical
a single
oxygen intermediates (RNI), and tissue
companions
production
addition of nitric oxide will increase production
whereas compounds
but ROl
IL-i
charide (LPS) will serve to induce continuing production of IL-8 protein and persisting levels of IL-8 mRNA in vitro [5]. This finding stands in contrast to most other cytokines, where the mRNA rapidly disappears [7], and the protein levels plateau after a few hours [8]. There is now ample
up-
the production blood, fibroblasts, cells,
and
Arbor
Ann
during inflammatory states. Therefore, cal basis and published data to support
Treatment as dimethyl
exogenous
School,
evidence
acute
will
antioxidants and do
hepatoma of
more serve
as initiators
other cytokines. scavengers such
production oxide
The hydroxyl pathway of
of
Addition
!L-8
of nitric
link sub-
destruc-
RO!
cells,
cytokines.
clear the
of pathogen
will decrease human whole
epithelial
other
a
and
gene expression since 8 (IL-8) production
decrease production with hydroxyl radical sulfoxide !L-8 in
also
Specifically,
cytokine interleukin
established cytokines
of ROt and RN!. However, that ROt and RN! not only
effector
inflammation. regulate
has
oxygen
Villarete
active nitrogen intermediates are frequent companions work production
by reactive
TNF,
ni-
reactive
sulfoxide;
superoxide
of
NO,
tumor
necrosis
dismutase;
CGD,
(L-N-nitroarginine
methyl
ester. Reprint requests: Daniel 1301 Catherine Road, Ann Received December
Journal
October 18,
30,
Remick, Arbor, 1995;
M.D., M2210 MI 48109-0602. revised
Sci 1, Box 0602,
Med
December
15,
1995;
accepted
1995.
of
Leukocyte
Biology
Volume
59,
April
1996
471
BIOCHEMISTRY AND RNI There
have
OF THE
been
several
FORMATION
studies
detailing
cytokines to induce ROl and RNI of cell types. Numerous cytokines to enhance IL-i,
ROl
IL-6,
and/or
IL-8,
peroxide
(H202),
several
compounds
pathways
capacity
of
from a variety demonstrated TNF,
factor. The metabolic intermediates and in formation and degradation of ROIs [ii, 18] and these pathways are shown
in Figure are the
cell
the
formation have been
production including and granulocyte-macrophage
interferon-y,
colony-stimulating enzymes important have been defined schematically within the
RN!
OF AOl
and
1. The superoxide
the
that
involved
major (02)
hydroxyl may
in RO!
radical
be used
formation,
ROl anion,
produced hydrogen
(OH).
There
are
to dissect
the
various
including
the
antioxi-
dant hydroxyl radical scavengers [dimethyl sulfoxide (DMSO), dimethyl thiourea, thiourea, etc.]. Another antioxidant defense mechanism is to maintain high intracellular levels of glutathione, which will effectively scavenge the
RO!. RNIs, the most (NO), are ultimately
widely studied formed from
of the highly NO synthases
reactive (NOS)
NO by the [19]. There
forms of NOS NO from the
identified terminal
to date. guanidino
of which is nitric oxide the enzymatic generation family of enzymes are three different
All three isoforms produce group of L-arginine. Two
of the isoforms are Ca2/calmodulin-dependent constitutively produced (ecNOS and ncNOS). the
third
isoform
(iNOS)
called iso-
and are In contrast,
physiological tein-bound
conditions, transition
metal
readily ions or
iron-nitrosyl
proteins
or
S-nitrosoproteins,
respectively.
these
have
a biological
NO
standpoint,
implicated to be responsible such diverse processes as tion,
platelet
inhibition,
1.
Metabolic
involved will
in the
block
their
pathways generation
of of ROl
production:
ROl
and
and
RNI,
(Reproduced
with
complexes thiol groups
with proforming
complexes
for the involvement neurotransmission,
host
defense,
and
been
of NO vasodilata-
in
inflammation.
IL-8,
LPS
and
stimulation
1.0 nglml (mean DMSO immediately
Our
THE
laboratory
cytokine inhibition
INDUCTION has
extensively
gene expression by of ROl will decrease
augmentation 23]. We have
OF CYTOKINES
significant ng/ml, P
mannitol or superoxide dismutase of IL-8. These results
catalase production since the hydrogen
mM)
listed from most in parenthesis):
thiourea
hydroxyl peroxide.
is intracellular
ethanol
>
(86
mM)
> DMSO
of ROI
that
drive
levels
enzymes amine (H202 oxamine whole binding We stimulated
of ROl to
will -
unless
improve
did
not
blood
reduce
system
the
reaction.
are
taken
of cytokine
This
we use,
may which
well has
gene
extended other cell
explored whole
the
blood
this enzyme is deficient lomatous disease (CGD) tients with CGD an up-regulation DMSO [22]. This through the action
IL-8 production. action of NADPH
in patients with [25]. When whole is stimulated
immune
ROIs
inhibitors.
xanthine Neither
complexes,
by of
human
cultured exhibited
cells to regula-
fibroblasts
were
incu-
which could this setting,
oxidase
in
the
is
which cause
generating
H2O2
be inhibited the glucose
extracellular
media,
will diffuse into the cell to up-regulate the ROl is generated outside of the cell, opportunity
to inactivate
the
H2O2
before
IL-B. catalase
Behas
it moves
into
their ability Fibroblasts,
to produce hepatoma
epithelial
cells
IL-B cells
(A549)
all secrete
when stimulated with 1% be inhibited by the addition Other
investigators
serve as publication
in response (HEP-G2)
to exogenous RO!. and type II alveolar
IL-B
into
H202, and of catalase
have
this
examined
intracellular second has supported our
the
supernatant
induction [22].
the
ability
could of ROl
messengers [28]. concept concerning
to
A recent the role
The -a-.
Glucose Oxidase (5mU/ml) Glucose oxidase + cotalse Catalase (100 U/mI)
--
-o--e-
the
Control
Deferoxreaction of defer-
be due very
of the
other
will inhibit of superoxide.
effectively decreased formed through the
phytohemagglutinin,
source
by using
in the
expression.
/
350O
/
to the
high
iron
/
capacity. further
human
was inhibitable to the importance
to isolated, types also
Isolated
the
to
increase
In
the cell intracel-
to modify [24].
prevent the Fenton however, addition
IL-8.
that
mM)
superoxide or states that it
bioavailability
chelate iron and OH) from occurring,
nol and oxypurinol prevent the formation hibitors is also
steps
their
potent to dimethyl (140
large catalase and SOD enzymes will not penetrate to achieve sufficiently high concentrations to alter lular
a substantial
and this increase results again point
by ROI.
increase
directly
the cell. This is in contrast to the LPS-stimulated whole blood, where catalase is not effective because the ROl are generated within the cell. Other cell lines were tested for
(50 mM). The addition of (SOD) did not block the may appear paradoxical,
radical is formed through However, our hypothesis levels
would
added
concentration in the supernatant, by the addition of catalase.
the using
superthat
bated in the presence of glucose oxidase (5 mU/ml) and the supernatants collected at different time points (Fig. 3). Glucose oxidase caused a substantial increase in the IL-8
0 IL-B
ROl
was
it caused
Our studies were determine whether of IL-8
exogenous H2O2
where
as regulators
tion
DMSO,
of IL-8.
of IL-8, [23]. These
ROI
20
was
of
amount DMSO
60
2.
as the source of the publications indicating
mitochondria may serve as a source of ROl [26, 27]. All of the data presented until this time have dealt with scavenging of ROl and decreasing IL-8. We also evaluated
100
Fig.
to the mitochondria is consistent with
in
/
iao
LPS-
Allopurioxidase of these
500
and in-
TT
Superoxide oxidase, and chronic blood with
or IL-i,
granufrom paLPS, there
is
of IL-8, which is inhibitable by 1% indicates that the ROI are not formed of NADPH oxidase. All of these data
0
4
8
12
16
20
24
TiME (hr
3.
Fig.
Kinetics
of glucose
broblasts.
Glucose
oxidae
broblasts
induces
the
prevented
by the
addition
are
means
catalase
± plus
(Adapted,
Remick
SE
for
and
permission,
Villarete
induction to the
synthesis
of
of catalase,
triplicate
glucose
with
oxidase added
wells.
oxidase
failed from
ROl
ref.
and
IL-8.
of lL-8
media This
which
will
No
stimulus,
to
induce
in
cultured
fi-
human
fi-
of cultured could
be
metabolize
completely H2O2.
catalase up-regulation
Values
alone,
and
of
IL-8.
22).
RNI regulate
cytokines
473
of ROI showing
in the induction that antioxidants
of chemokine will also
monocyte chemotactic line U937, antioxidants TNF
[30].
protein will
In contrast,
our
expression with depress production
data of
In the monocyte the up-regulation
cell of
[29]. prevent
studies
did
not
show
any
cant inhibition of TNF using stimulated whole differences probably relate to the experimental since
the
enging
whole
blood
capacity.
tory have shown human mononuclear
that
(unpublished data). induced by exposure duce
IL-8
substantial
oxidant
regard,
experiments
in our
the cells
human
mononuclear
work with fibroblasts failed induce IL-8 [22]. Additional that
steps
will this
inhibit setting,
the
intracellular
prevent
to maintain
the
stores
scav-
to show studies
levels
of glutathione
accumulation
8) 0
40
20
Our
that cadmium in vivo have
intracellular
would shown IL-fl
of glutathione
are
not
[32]. In LPS) and
sufficient
to
of ROIs.
Fig.
4.
whole
Our
studies
OH that
on IL-B production RNIs may also exert
demonstrating
the
L-NAME,
Blockade
of NO
regulatory
formation to parallel
because signaling
NO has as well
(Fig. 1) [19]. These experiments were those used in examining the regulation
of cytokine gene expression by ROIs. The availability of NO synthase inhibitors and NO-generating compounds have made it possible to evaluate the effect of NO in regulating cytokine gene expression. Thus far, our studies have provided ample evidence substantiating an immunoregulatory role for blood system
NO in IL-B production and in the human
in the endothelial
human whole cell line,
ECV3O4.
Figure
4 depicts a representative set showing the LPS (1 tg/ml) -induced
donor
IL-B (18.8 ng/ml) L-N-nitroarginine inhibitor,
to blood
2 h before
decrease
This
effect
LPS
in IL-B
stimulation
but
of L-NAME
not IL-6
was
also occurred at the mRNA level. The synthase inhibitors have the potential duction human inducer
resulted protein
dose-dependent observation to inhibit
in
levels. and that NO IL-B pro-
in human whole blood has been extended to the endothelial cell line, ECV3O4. TNF is a potent of IL-B in endothelial cells [33]. Stimulation of
ECV3O4
cells
levels
with
of IL-8,
manner The
50
which
by the addition next series of
whether production
474
from one in both
and IL-6 (87.8 ng/ml). Addition of 5mM methyl ester (L-NAME), a NO synthase
the substantial inhibitory
of data increase
nglml was
of TNF
inhibited
of L-NAME experiments
induced
significant
in a dose-dependent [34]. was
used
to evaluate
exogenously added NO could stimulate cytokine in the whole blood model or the ECV3O4 cells.
Journal
of
Leukocyte
Biology
Volume
59,
April
1996
with cytokines resulted
in the
amount
of >15 L-NAME
IL-8
in
the
of IL-6.
production.
plasma Data
donors.
a significant
Human
in the presence
or absence
measured
in a reduction
individual
causes
compared
decreases 1 I.lgf ml of LPS
in the are
from
When
24 amount
a single the
inhibition
donor
groups
of IL-8
h later. of IL-8, are
but com-
production.
to LPS.
of
to explore the possibility effects on cytokine pro-
duction. We explored this hypothesis been shown to be involved in intracellular as OH designed
effect
5 mM 0.05
synthase
and
no reduction
*P
