Extracellular superoxide dismutase is upregulated with
inducible nitric oxide synthase after NF-
B activation
Todd C.
Brady1,
Ling-Yi
Chang2,
Brian J.
Day2, and
James D.
Crapo2
1 Department of Pathology,
Program in Integrated Toxicology, Duke University Medical Center,
Durham, North Carolina 27710; and
2 Department of Medicine,
National Jewish Medical and Research Center, Denver, Colorado 80206
 |
ABSTRACT |
Inflammatory cytokines have been shown to
upregulate secretion of the antioxidant enzyme extracellular superoxide
dismutase (EC-SOD) in dermal fibroblasts and, in other cells, to
stimulate production of nitric oxide (· NO). Because
superoxide rapidly scavenges · NO, forming the injurious
peroxynitrite anion
(OONO
), we hypothesize
that stimulated cells upregulate EC-SOD expression concurrently with
· NO release. To test for coregulation of EC-SOD and
· NO within the same cell, the timing of inducible nitric oxide synthase (iNOS) and EC-SOD transcription was measured after exposure of a rat type II pneumocyte analog, the L2 cell line, to a
combination of interferon-
(IFN-
) and tumor necrosis factor-
(TNF-
). Upregulation of iNOS and EC-SOD transcription occurred after
6 h of exposure, and transcription of both genes was linked by
activation of the transcription factor nuclear factor-
B. Both EC-SOD
and iNOS were elevated in rat lung homogenates 24 h after intratracheal
instillation with IFN-
and TNF-
. The observation that EC-SOD and
iNOS are temporally coregulated after cytokine exposure suggests the
possibility of a critical mechanism by which cells might protect
· NO and avoid the formation of
OONO
during inflammation.
interferon-
; tumor necrosis factor-
; alveolus; lung; nuclear
factor-
B
 |
INTRODUCTION |
EXTRACELLULAR SUPEROXIDE DISMUTASE (EC-SOD) is a
secreted antioxidant enzyme especially prevalent in lungs (12) and
functions to protect cells and connective tissue from extracellular
superoxide (
). The dismutation
of
is important not only
because
itself is a damaging
radical but also because
can
rapidly react with nitric oxide (· NO) to form the
peroxynitrite anion (OONO
;
see Refs. 19 and 20)
At
physiological pH, OONO
decomposes into intermediates with reactivities similar to the hydroxyl
radical (HO ·) and NO2 (2)
Thus,
by scavenging
, EC-SOD may
protect · NO outside the cell. Because EC-SOD binds to the
cell membrane and proteoglycans in the extracellular matrix (8), the
enzyme may reduce the likelihood of extracellular
OONO
, HO ·, and
· NO2 formation near
plasma membranes or connective tissue during · NO release.
In environments in which increased amounts of
and · NO are
produced during inflammation, such as in the alveolus, the role of
EC-SOD in protecting · NO bioavailability and preventing
OONO
formation may be
critical. Under basal conditions, in situ hybridization studies have
demonstrated the presence of EC-SOD mRNA in a secretory alveolar
epithelial cell known as the type II cell (23). Although transcription
of EC-SOD after cytokine treatment has not been characterized in any
cell, exposure to combinations of the inflammatory cytokines
interferon-
(IFN-
) and tumor necrosis factor-
(TNF-
) has
been shown to increase EC-SOD secretion in dermal fibroblasts (13).
After treatment with other combinations of cytokines in different
scenarios, type II pneumocytes in culture express inducible nitric
oxide synthase (iNOS; see Ref. 18), an · NO-producing enzyme
synthesized in response to environmental stimuli. Because
rapidly scavenges
· NO, forming
OONO
and other reactive
species, we hypothesize that · NO-secreting cells such as
type II pneumocytes upregulate EC-SOD expression before or concurrently
with · NO release. To test for coregulation of EC-SOD and
· NO within the same cell, the timing of EC-SOD transcription
was compared with that of iNOS in a rat type II pneumocyte analog, the
L2 cell line, after treatment with IFN-
and TNF-
. Because no
functional transcription factors for EC-SOD have been demonstrated, we
examined the relationship between EC-SOD transcription and the
activation of nuclear factor-
B (NF-
B), a transcription factor
known to upregulate iNOS transcription (17, 26), to link the initiation
of EC-SOD and iNOS transcription. Temporal changes in media EC-SOD and
nitrite levels were also compared after cytokine treatment.
Other studies have demonstrated the presence of EC-SOD in lungs under
basal conditions (12) and iNOS in stimulated lungs (7, 10). To test for
concurrent upregulation of EC-SOD and iNOS expression in lung, IFN-
and TNF-
were intratracheally instilled in rats, and protein levels
were measured in lung homogenates. We present evidence characterizing
the timing and NF-
B-mediated control of stimulated EC-SOD
transcription and thereby establish the temporal relationship between
EC-SOD and · NO production within the same cell and animal
models of inflammation.
 |
EXPERIMENTAL PROCEDURES |
Cell and animal exposure. L2 cells
were purchased from the American Type Culture Collection (CCL-149;
Rockville, MD) and were grown in Ham's F-12-K (Kaighn's modification)
media (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine
serum (FBS; GIBCO), penicillin (10 U/ml), and streptomycin (10 mg/ml;
GIBCO). Cells were grown in 5%
CO2 at 37°C and were used
between the 40th and 45th passages. For transcription analysis,
~105 cells were plated in 2 ml
of media and were grown to confluency in individual 35-mm dishes
(Falcon, Lincoln Park, NJ). At confluency, media was replaced with 1 ml
of F-12-K containing 1% FBS. Twenty-four hours after confluency was
attained, cytokine exposures were initiated. Murine IFN-
(2,000 U/ml; Genzyme, Cambridge, MA) and TNF-
(500 U/ml; a gift from Dr.
Grace Huang, Gentech), both of which have been shown to stimulate rat
cells (4, 14, 24), were added to 1 ml of 1% FBS media either with or
without 100 µM pyrrolidinedithiocarbamate (PDTC; Sigma, St. Louis,
MO; see Refs. 1 and 9). Various times (3, 6, 12, and 18 h) after
cytokine exposure, medium was removed and was stored at
70°C
until needed for nitrite determinations or EC-SOD Western blots. Cells
were then lysed for RNA extraction. Eight male Sprague-Dawley rats (350 g; Charles River Laboratories, Wilmington, MA) were intratracheally
instilled with 500 µl of 0.9% NaCl, either with or without 100,000 U/ml IFN-
and 25,000 U/ml TNF-
. Twenty-four hours after
instillation, rats were killed with 100 mg/kg phenobarbital sodium, a
thoracotomy was performed, and the middle lobe of the right lung was
excised.
RNA isolation and reverse transcriptase-polymerase
chain reaction. Pure RNA was isolated by phenol
extraction from a guanidium thiocyanate lysis solution (Biotecx,
Houston, TX). Immediately after cell lysis, the guanidium solution was
frozen and was stored at
70°C. After RNA isolation, reverse
transcriptase-polymerase chain reaction (RT-PCR) was
performed using recombinant Tth polymerase with magnesium
and manganese acetate buffers (Perkin-Elmer, Foster City, CA). Novel
primers were created for rat EC-SOD (5'-TAG CCT AGC TGC TGC GCG
CAT A; 3'-GGG CGC ACA GAG GCG ATT GA) and rat iNOS (5'-AGC
ACA TGC AGA ATG AGT ACC; 3'-TGA TGC TCC CGG ACA CCG GA). RT-PCR
for both EC-SOD and iNOS tubes were run simultaneously under the same
thermocycling protocol: 30 min at 60°C × 1 cycle; 3 min at
94°C × 1 cycle; 20 s at 94°C followed by 30 s at 70°C × 40 cycles; and 7 min at 70°C × 1 cycle. RT-PCR
samples were run in 3% agarose and were stained with ethidium bromide.
Both iNOS and EC-SOD primers produced unique bands of 83 and 123 base pairs, respectively. Control RT-PCR reactions for rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were run
separately using novel primers (5': GGT GTC AAC GGA TTT GGC CGT
ATT; 3': CAT GCC AGT GAG CTT CCC GTT CA), yielding a unique band
of 674 base pairs.
Western blotting. Thawed media were
spun at 5,000 g for 30 min at 4°C
in Centricon-30 concentrators (Amicon, Beverly, MA) to yield a protein
concentration of ~125 mg/ml. Lung tissue was homogenized in a
mechanical homogenizer in ice-cold lysis solution [50 mM
tris(hydroxymethyl)aminomethane · HCl, pH
7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM
ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotonin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4,
and 1 mM NaF]. Lung homogenate was centrifuged at 14,000 revolutions/min for 10 min at 4°C, and the supernatant was stored at
70°C. Approximately 2,500 mg of cell media protein or 50 mg of lung homogenate supernatant protein were loaded on a 12%
polyacrylamide gel in 2-amino-2-methyl-1,3-propanediol
buffers and electrophoresed on a Minigel apparatus
(Hoefer, San Francisco, CA). Western blotting for EC-SOD was performed
using a polyclonal rabbit antibody against a murine EC-SOD peptide
previously sequenced (3). Blotting for iNOS was performed using a
polyclonal rabbit antibody against murine iNOS (Transduction
Laboratories, Lexington, KY). Chemiluminescent detection was performed
using a horseradish peroxidase-conjugated secondary antibody (ECL
System, Amersham, Buckinghamshire, UK).
Nitrite determinations. For nitrite
determinations, growth conditions were the same as those described
above except that 35-mm six-well plates (Falcon) were used instead of
individual dishes. At different times after exposure, nitrite in the
media of L2 cells stimulated with IFN-
and TNF-
was detected
using the Griess reagent and sodium nitrite as the standard, which has
been described elsewhere (6). Nitrite determinations for each condition
were performed in triplicate. Cytokine-treated wells were compared with
control (nonexposed) wells at various times (12, 18, 24, and 30 h)
after cytokine exposure.
Statistics. Multiple-way analyses of
variance (ANOVAs) followed by one-degree-of-freedom contrasts of means
(
= 0.05) were used to compare nitrite concentrations of
cytokine-treated wells and control wells at all times after exposure.
The means of nitrite concentrations in control wells were contrasted
across time via ANOVA followed by Duncan's multiple range test.
 |
RESULTS |
Nitrite and EC-SOD in L2 cell media are increased
after cytokine exposure. L2 cells were exposed to a
combination of IFN-
and TNF-
in an attempt to compare the timing
of EC-SOD and · NO production within the same cell. Nitrite,
which is a stable product of · NO and generally correlates
with the release of · NO, was detected via the Griess reagent
method (6) in the media of control cells and cytokine-exposed cells. At
all times after exposure, the media of wells exposed to cytokines had
greater amounts of nitrite than control wells (Fig.
1). These differences were statistically
significant by 24 and 30 h after exposure. Nitrite did not increase
significantly in control wells over time. Western blot analysis of the
media from exposed cells demonstrated EC-SOD in the media at 24 and 36 h after the addition of IFN-
and TNF-
. Greater EC-SOD labeling
was evident at 36 h. Media from cells not exposed to cytokines did not
label for EC-SOD at incubation times up to 24 h (data not shown).
Although rat and mouse EC-SOD protein are ~80% homologous (3, 25),
lack of basal EC-SOD labeling in the media of control cells and at
times before 24 h postexposure may be related to the use of an antibody against murine EC-SOD to detect rat protein.

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Fig. 1.
Cytokine treatment increases extracellular superoxide dismutase
(EC-SOD) and nitrite in L2 cell media. L2 cell media were withdrawn at
various times after exposure to interferon- (IFN- ) and tumor
necrosis factor- (TNF- ). Nitrite in the media was measured via
the Griess reagent (6). Exposed wells significantly differed from
control wells by 24 and 30 h after exposure, as indicated by asterisks
(n = 3/condition,
P < 0.01). No significant
differences were evident between controls at any time. Concentrated
media protein was electrophoresed and blotted with an antibody against
murine EC-SOD; 0 h represents media not placed on cells. At the
expected size of 36 kDa (25), a band of increasing intensity is noted
at 24 and 36 h after cytokine exposure.
|
|
iNOS and EC-SOD transcription are upregulated in L2
cells after cytokine exposure. After exposure to
IFN-
and TNF-
, L2 cell transcription of iNOS and EC-SOD mRNA was
studied by RT-PCR. No iNOS mRNA was detected before cytokine exposure,
but iNOS signal was evident by 6 h after cytokines were added (Fig.
2). Basal levels of EC-SOD mRNA in L2 cells
were present before cytokine treatment, consistent with previous in
situ studies with type II pneumocytes (23). After exposure to IFN-
and TNF-
, EC-SOD signal increased, indicating transcriptional
upregulation from basal levels. Upregulation of EC-SOD signal continued
through 18 h after exposure to cytokines. No differences were observed between GAPDH mRNA amplified from control and exposed L2 cells at all
times after exposure (data not shown). These studies showed that
IFN-
and TNF-
initiated iNOS transcription and upregulated EC-SOD
transcription in L2 cells by 6 h after exposure, confirming the
increases in media levels of nitrite and EC-SOD observed after cytokine
treatment. The data suggest that upregulation of EC-SOD transcription
may occur slightly before that of iNOS.

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Fig. 2.
Cytokine exposure upregulates inducible nitric oxide synthase (iNOS)
and EC-SOD transcription in L2 cells. Reverse transcriptase-polymerase
chain reaction (RT-PCR) was performed using RNA isolated from L2 cells
at various times after exposure to IFN- and TNF- . No exposure is
represented by time 0. Note that no
iNOS is transcribed before cytokine stimulation but that EC-SOD is
transcribed constitutively. iNOS transcription is evident from 6 to 18 h after exposure. EC-SOD transcription is increased through 18 h after
stimulation.
|
|
iNOS and EC-SOD transcription are regulated by
NF-
B in cytokine-exposed L2 cells. iNOS
transcription is initiated by activation of the transcription factor
NF-
B (17, 26). The metal chelator and antioxidant PDTC has been used
to inhibit NF-
B activation but does not inhibit activity of other
transcription factors, such as adenosine 3',5'-cyclic
monophosphate response element binding protein, activator protein-1,
octamer-1, and Sp1 (21). PDTC treatment prevents
cytokine-mediated induction of iNOS in a variety of cells (1, 9). As
expected, addition of PDTC with cytokines to L2 cells blocked IFN-
-
and TNF-
-induced iNOS transcription (data not shown). PDTC
and cytokine treatment resulted in EC-SOD transcription only at basal
levels (Fig. 3). Because PDTC is an
inhibitor of NF-
B activation, these data suggest that the
upregulation of EC-SOD transcription and the initiation of iNOS
transcription in response to IFN-
and TNF-
are both controlled by
NF-
B in L2 cells. Such regulation further supports the coexpression of EC-SOD and iNOS in this model.

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Fig. 3.
Pyrrolidinedithiocarbamate (PDTC) treatment prevents cytokine-mediated
upregulation of EC-SOD transcription in L2 cells. RT-PCR was performed
using RNA extracted from L2 cells exposed to IFN- and TNF- either
with 100 µM PDTC (PDTC + Cyto) or without PDTC (Cyto). RNA was
extracted at 3 and 6 h after exposure. Lane labeled control indicates
no exposure. Note that signal in PDTC lanes is equivalent to that of
the control, indicating that cytokines did not upregulate EC-SOD in
PDTC-treated dishes and that PDTC treatment did not decrease
constitutive EC-SOD transcription. EC-SOD transcriptional upregulation
is clearly seen in cytokine-treated cells without PDTC.
|
|
iNOS and EC-SOD levels are increased in rat lung after
cytokine treatment. Expression of iNOS and EC-SOD
protein was studied in rat lung 24 h after intratracheal instillation
of IFN-
and TNF-
and was compared with saline-instilled rats
(n = 4/condition). Western blotting
indicated the presence of iNOS only in cytokine-instilled lungs (Fig.
4). EC-SOD expression was higher in the
exposed lungs compared with saline-instilled lungs. These data are
consistent with the increase in EC-SOD and iNOS expression observed in
cytokine-treated L2 cells.

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Fig. 4.
iNOS and EC-SOD expression is upregulated in cytokine-instilled rat
lung. Rats were intratracheally instilled with or without 100,000 U/ml
IFN- and 25,000 U/ml TNF- in 500 ml of saline. Twenty-four hours
after instillation, the middle lobe of the right lung was removed and
homogenized for Western blot analysis. There was no detectable iNOS
protein in the saline-treated rats. However, basal levels of EC-SOD
were present in control rat lung. Cytokine-treated rat lungs labeled
strongly for iNOS and manifested increased levels of EC-SOD.
|
|
 |
DISCUSSION |
This report presents data demonstrating the control of EC-SOD
transcription by cytokines and the temporal correlation of iNOS and
EC-SOD expression after cytokine exposure within the same cell and
animal models. RT-PCR, Western blotting, and nitrite analysis of
IFN-
- and TNF-
-stimulated L2 cells suggest that, during
inflammation, when IFN-
and TNF-
are likely to be present, EC-SOD
transcription and secretion are increased above basal levels concurrently with, or perhaps slightly before, upregulation of iNOS
transcription and · NO release. Induction of EC-SOD secretion and · NO release is linked by activation of NF-
B, which
simultaneously upregulates transcription of EC-SOD and iNOS. The whole
lung data indicate that IFN-
and TNF-
instillation increases both
iNOS and EC-SOD expression in rat lung and are consistent with the L2
cell results. We suggest that upregulation of iNOS and EC-SOD expression occurs in the same vicinities within the lung and at approximately the same time. Further characterization of the
relationship between EC-SOD and iNOS expression in response to either
IFN-
or TNF-
alone or to other cytokines present in different
biological scenarios, both in cell culture and in situ, merits study.
Numerous transcription factors regulating iNOS have been characterized
and include NF-
B, TNF responsive element, and IFN responsive element
(17). NF-
B has been shown to be necessary for iNOS transcription in
various cells (1, 9). Our results with L2 cells are consistent with
these studies, since PDTC, an inhibitor of NF-
B activation,
prevented transcription of iNOS. No study has previously demonstrated a
functional transcription factor regulating expression of EC-SOD. This
report demonstrates that PDTC also prevents upregulation of EC-SOD
transcription but does not inhibit basal EC-SOD transcription,
implicating activation of NF-
B in the induction of EC-SOD
expression. The result indicates a potential mechanism for the observed
cytokine-mediated coregulation of EC-SOD and iNOS expression,
suggesting that IFN-
and TNF-
increase EC-SOD and iNOS
transcription in L2 cells via activation of NF-
B. Activated NF-
B
may act directly by stimulating EC-SOD transcription or indirectly by
influencing different factors that, in turn, lead to upregulation of
EC-SOD. Although other transcription factors may influence EC-SOD and
iNOS in L2 cells, activation of NF-
B appears to be necessary for
transcription of iNOS and for increased EC-SOD transcription after
exposure to IFN-
and TNF-
. Because normal levels of EC-SOD
transcription were evident during PDTC treatment, it is unlikely that
PDTC prevented iNOS transcription or EC-SOD upregulation by a
nonspecific response to generalized cell injury. PDTC inhibits NF-
B
activation but does not affect the function of other transcription
factors (21). Nonetheless, the possibility exists that the compound may
diminish upregulation of EC-SOD transcription by means not associated
with NF-
B activation, including acting as an antioxidant, chelating metals, or affecting other transcription factors yet to be
characterized.
In the stimulated L2 cell and whole lung models, the finding that
EC-SOD and iNOS upregulation are temporally linked after activation of
NF-
B suggests that major amounts of · NO do not cross the
plasma membrane before EC-SOD secretion. If EC-SOD upregulation occurred late in the inflammatory response after significant
· NO release in the presence of extracellular
, then the cell might suffer
damage from
as well as from
radicals derived from OONO
.
Because EC-SOD binds to cell membranes and the extracellular matrix
(8), released · NO may be protected from
only within these areas.
Further experiments will elucidate the effects of · NO
release in inflammatory environments and verify the necessity of
protecting · NO and preventing
OONO
formation near cells
and associated connective tissue during inflammation. · NO
may act on targets within the zone protected by EC-SOD to exert muscle
relaxation (16), act as a signaling molecule (5), or modulate immune
responses of nearby inflammatory cells to prevent host injury (15, 22).
Alternatively, · NO may diffuse from the cell and associated
extracellular matrix (e.g., into the alveolar space) to react with
, forming
OONO
and other injurious
compounds, perhaps targeting invading organisms (11). Thus coregulation
of EC-SOD and iNOS may be a critical cellular response during
inflammation. By synchronizing the expression of these enzymes, the
cell may protect itself and the surrounding matrix from extracellular
,
OONO
, HO ·, and
· NO2 while increasing
the efficacy of secreted · NO as a functional effector
outside the plasma membrane.
 |
ACKNOWLEDGEMENTS |
This work has been supported by National Institutes of Health
Grants T32-ES-07041-17, RO1 HL-42609, and PO1 HL-31992.
 |
FOOTNOTES |
Address for reprint requests: J. D. Crapo, Dept. of Medicine, National
Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206.
Received 8 May 1997; accepted in final form 31 July 1997.
 |
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