Departments of 1 Internal Medicine, 2 Molecular Microbiology and Immunology, and 3 Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63110-0250
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ABSTRACT |
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Episodes
of tissue hypoxia and reoxygenation frequently occur during
gram-negative bacteremia that progresses to septic shock. However, few
studies have evaluated modulation by hypoxia and reoxygenation of the
proinflammatory cytokine gene expression that is normally induced by
gram-negative bacteremia or endotoxemia. In buffer-perfused organs,
hypoxia downregulates Escherichia coli-induced expression of
tumor necrosis factor (TNF)- and interleukin (IL)-1
in the liver
but upregulates these cytokines in the lungs. To identify molecular
mechanisms underlying these events, we investigated the effects of
brief (1.5-h) hypoxia on TNF-
and IL-1
expression in cultured RAW
264.7 cells during their continuous exposure to lipopolysaccharide
(LPS) endotoxin derived from E. coli (serotype 055:B5) for up
to 24 h. IL-1
and TNF-
concentrations in cell lysates and culture
supernatants were measured by ELISA, and steady-state mRNA was measured
by Northern analysis. LPS-induced IL-1
synthesis was downregulated
by hypoxia at both the protein and mRNA levels despite no change in
cellular redox status as measured by levels of GSH. In contrast,
LPS-induced TNF-
production was unaffected by hypoxia as assessed by
cell lysate mRNA and lysate and supernatant protein levels. Nuclear
runoff analysis showed that downregulation of
IL-1
gene expression by hypoxia occurred
transcriptionally. Allopurinol or catalase treatment did not alter
modulation of LPS-induced IL-1
expression by hypoxia, suggesting
that this suppression was not caused by reactive oxygen species.
Cycloheximide pretreatment suggested that hypoxia-induced
downregulation of IL-1
expression did not require de novo protein synthesis.
lipopolysaccharide; interleukin-1; tumor necrosis factor-
; septic shock; endotoxin; redox status; cytokines; inflammation; acute
respiratory distress syndrome; organ failure; reactive oxygen species
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INTRODUCTION |
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DURING GRAM-NEGATIVE BACTEREMIA (GNB) or endotoxemia,
the inappropriate regulation of lipopolysaccharide (LPS)-induced
inflammatory responses often culminates in shock and progressive injury
to multiple organ systems (4, 22). Among host defense tissues, the
liver and lungs have emerged as key regulatory organs that modulate
innate responses to GNB and LPS through their production of pleiotropic
cytokines, notably interleukin (IL)-1 and tumor necrosis factor
(TNF)-
(22). In addition to causing local injury, hepatic and
pulmonary export of these early proinflammatory mediators induces
cytokine-dependent increases in microvascular permeability and
neutrophilic influx that typify the acute respiratory distress syndrome
and the multiple-organ dysfunction syndrome (18, 22). Thus
understanding the regulation of host proinflammatory cytokine expression during conditions that may precipitate shock is vital to
developing rational treatments for septic patients.
Two major features of the innate host inflammatory responses to LPS and
GNB that may precipitate acute respiratory distress syndrome and
multiple-organ dysfunction syndrome have emerged. First, overexpression
of IL-1 and TNF-
will be further amplified by their induction of
secondary mediator pathways, particularly nitric oxide from inducible
nitric oxide synthase and cyclooxygenase-2 (7, 16, 24). Second,
reactive oxygen species (ROS) that are generated by exposure to LPS or
GNB and that directly stimulate cytokine expression (19, 20, 26) may
themselves be altered by episodes of tissue hypoxia and reoxygenation
(H/R) that commonly occur during shock or other trauma (26, 28, 30). Of
note, our laboratory recently showed that brief
postbacteremic H/R blunted the Escherichia coli-induced
expression of TNF-
, IL-1
, and IL-1
at the mRNA and protein
levels in buffer-perfused rat livers (23, 30), but H/R enhanced their
expression in buffer-perfused rat lungs (25). We also found that
xanthine oxidase (XO)-derived ROS were critical to the H/R-induced
downregulation of IL-1
but not of TNF-
and IL-1
expression by
the liver by using the XO substrate inhibitor allopurinol (Allo) (23,
30).
The present study was undertaken to better characterize the basic
mechanisms of hypoxia-induced suppression of cytokine synthesis. We
chose the RAW 264.7 murine monocyte-derived cell culture model in which
cytokine expression has been extensively characterized (1, 2, 10).
LPS-treated RAW 264.7 cells were exposed to 1.5 h of hypoxia and
assessed for their IL-1 and
TNF-
gene expression for up to 24 h. As in
perfused livers, this brief hypoxia reversibly suppressed IL-1
synthesis at the mRNA and protein levels. Nuclear runoff analysis
indicated that hypoxic suppression occurred at the level of
transcription. Furthermore, the effects of hypoxia could not be
prevented with Allo, catalase, or cycloheximide (CHX), suggesting that
neither ROS nor de novo protein synthesis was required for this
downregulation. These effects on IL-1
expression occurred despite no
change in intracellular GSH concentration ([GSH]) due to
hypoxia. Surprisingly, LPS-induced increases in TNF-
steady-state
mRNA, protein, and transcription rates were not affected by hypoxia.
Taken together, the results indicate that brief hypoxia reversibly
decreases LPS-induced IL-1
synthesis in RAW 264.7 cells, at least in
part by reducing their transcription rate of this canonical
proinflammatory cytokine.
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METHODS |
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Reagents. Fetal bovine serum (FBS) was from HyClone (Logan, UT); and high-glucose Dulbecco's minimum Eagle's medium (DMEM), L-glutamine, penicillin, and streptomycin were from GIBCO BRL (Life Technologies, Grand Island, NY). Reagents for molecular biological studies included agarose from Amresco (Solon, OH) and RNase I and DNase from Promega (Madison, WI). Purified LPS derived from E. coli serotype 055:B5, Allo, Tris, sodium dodecyl sulfate (SDS), Triton X-100, Tween 20, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, proteinase K, and other tissue culture-grade chemicals were from Sigma (St. Louis, MO). Guanidine thiocyanate, HEPES, dithiothreitol, MOPS, phenol, creatine phosphate, unlabeled deoxynucleotide triphosphate, and creatine phosphokinase were also from Sigma. The 32P-radiolabeled nucleotides dUTP and dCTP were from Amersham Life Science (Arlington Heights, IL). Denhardt's solution was from Boehringer Mannheim (Indianapolis, IN), and anhydrous alcohol for RNA and DNA extractions was from Eastman Kodak (Rochester, NY).
Cell culture and experimental protocol. Mycoplasma-free RAW 264.7 cells were cultured at a seeding density of 5 × 105 cells/well in 24-well microtiter plates or in 50- or 100-mm plates with 5 × 106 or 1 × 107 cells/well, respectively, for Northern hybridization or nuclear run-on assays in DMEM with 5% heat-inactivated FBS, 1% L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin and grown to confluence (37°C for 48 h). Culture medium was replaced with fresh DMEM at the start of experiments, after which baseline supernatant samples were collected 0.5 h later [time (t) = 0 h]. RAW 264.7 cell monolayers were then immediately stimulated with DMEM containing LPS (100 ng/ml) for up to 24 h. Preliminary experiments with a higher dose of LPS (1 µg/ml) gave similar results and were not pursued in this study. As negative controls, isovolumetric DMEM alone was added in other cultures.
Normoxic LPS-stimulated cells as well as the LPS-free DMEM control cultures were continuously incubated in a 21% O2-5% CO2-74% N2 atmosphere for up to 24 h. In parallel cultures, secondary hypoxic stress was induced by switching to a 95% N2-5% CO2 incubating gas mixture in a controlled atmospheric chamber (Billups-Rothenberg, Delmar, CA) for 1.5 h starting 0.5 h after the addition of LPS or DMEM (i.e., t = 0.5 h). Four major experimental groups were studied in quadruplicate cultures: normoxic LPS control; LPS plus hypoxia with and without reoxygenation (H/R) that began at t = 2.0 h; DMEM normoxic control; and DMEM plus H/R. Hypoxic exposure was confirmed in each experiment by measuring the ambient PO2 of the gas above the monolayers (IL-1306 blood gas analyzer, Instrumentation Labs, Lexington, MA), which averaged 10 ± 1 (SE) mmHg within 5 min of hypoxia and remained stable through t = 2.0 h compared with a normoxic level of 140 ± 2 mmHg. Corresponding reductions in the liquid-phase PO2 values were indicated by medium PO2 values of 42 ± 2 mmHg throughout hypoxia compared with a time-matched normoxic level of 142 ± 2 mmHg. Reoxygenation of hypoxic cultures was achieved with 21% O2-5% CO2-74% N2, with ambient and liquid-phase PO2 values returning to prehypoxic levels within 5 min. Supernatants of cells cultured in normoxia, hypoxia, and H/R were collected, and the cells were lysed at t = 0, 0.5, 2, 3, 6, and 24 h. Supernatants and lysates that were processed as described below were stored atImmunoreactive IL-1 and TNF-
analyses.
Immunoreactive IL-1
in supernatants and cell lysates was determined
in duplicate by an ELISA sensitive over a 0-960 pg/ml range
(BioSource International, Camarillo, CA) (19, 23). Reactions used a
monoclonal anti-IL-1
capture antibody and a biotinylated polyclonal
anti-murine IL-1
antibody and were developed with streptavidin-horseradish peroxidase with absorbance measured at 450 nm.
For cell-associated cytokine concentrations, the lysates were chilled
to 0°C for 20 min, centrifuged (14,000 g for 15 min), and
diluted 1:50 in phosphate-buffered 0.9% NaCl (PBS). Total protein
concentrations were measured in lysates with the bicinchoninic method
(Pierce, Rockford, IL), with the results expressed in nanograms of
IL-1
per milligram of cellular protein. Supernatant and lysate concentrations of immunoreactive TNF-
were similarly determined in
duplicate with a solid-phase ELISA (17, 25). This assay was sensitive
to murine TNF-
over a range of 50-3,200 pg/ml with a rabbit
anti-murine anti-TNF-
and a goat anti-rabbit IgG linked to
horseradish peroxidase, followed by analysis at 450 nm (Bio-Tek EL-311,
Winooski, VT). Recombinant murine TNF-
and the primary and secondary
antibodies were obtained from Genzyme (Cambridge, MA). Interassay
coefficients of variation for IL-1
and TNF-
determinations were
8.0 and 1.4%, respectively.
Northern hybridizations.
Total RNA was isolated from a minimum of 5 × 106
cells with the method of Chomczynski and Sacchi (5) with modifications (23, 30). Briefly, the cells were lysed in RNA STAT-60 reagent, precipitated with isopropanol, and washed once with 75% ethanol. The
resulting RNA was quantitated by optical density, and 20-µg samples
were resolved by electrophoresis on 1.5% agarose gels containing 1.1 M
formaldehyde. Gels were run for 2-4 h at 75 V in 1× MOPS (pH
7.0) running buffer. RNA on the gels was transferred directly to nylon
membranes, with hybridization performed at 68°C for 1 h. A 1,100-bp
cDNA for murine TNF-, a 1,301-bp cDNA for murine IL-1
, and a
960-bp murine cDNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
were random-primer labeled with 32P as previously described
(23, 30). After hybridization, the membranes were washed at
65°C before autoradiography was performed at
70°C.
Steady-state mRNA levels were measured for at least three replicates
per treatment group and per time point by densitometry in the linear
range of detection (Molecular Dynamics, Sunnyvale, CA), with results
normalized for differences in loading with the GAPDH signals.
Nuclear runoff experiments.
LPS-stimulated cells exposed to normoxia or hypoxia with and without
reoxygenation were lysed for analysis of transcriptional activity of
the IL-1, IL-
, and
TNF-
genes, with pBluescript serving as a negative
control and GAPDH as loading control. Freshly isolated
nuclei from ~2.5 × 107 cells in each treatment
group were obtained as previously described (1, 29). Cells were scraped
from 100-mm plates, washed once in 4°C PBS, and lysed in buffer
containing 10 mM HEPES (pH 8.0), 10 mM KCl, and 1.5 mM
MgCl2, followed by repeated passage through a
25-gauge needle.
Intracellular GSH. Cellular concentrations of GSH, a sensitive indicator of induced oxidative stress, were measured in cell lysates by HPLC separation as previously described (26, 30). Results are expressed as micromoles of GSH per milligram of cellular protein.
Lactate dehydrogenase assay. The effects of secondary hypoxia alone and of H/R on RAW 264.7 cell viability in LPS-stimulated and DMEM control cultures were assessed in quadruplicate. Supernatants were evaluated for their lactate dehydrogenase concentration with a standard kit assay (procedure 228-UV, Promega).
Direct measurement of ROS formation in RAW 264.7 cells. For these experiments, RAW 264.7 cells were cultured at a seeding density of 5 × 105 cells/well in 24-well microtiter plates in DMEM supplemented with 5% heat-inactivated FBS and 1% L-glutamine and grown to confluence overnight. The supernatants were replaced with fresh DMEM containing 10 µM 2',7'-dichlorofluorescein diacetate dye as probe and were incubated in the dark for 15 min (26, 28). The cells were then washed with fresh medium to remove excess dye and cultured with either DMEM containing LPS (100 ng/ml) or DMEM alone under both normoxic and hypoxic conditions as described in Cell culture and experimental protocol. At peak hypoxia (t = 2.0 h), the medium was removed and PBS was added, followed by harvesting of the cells for determination of fluorescence emission at 525 nm (Aminco/Bowman Luminescence Spectrometer, Urbana, IL). Dye-loaded cells that were incubated for 15 min in the presence of H2O2 (10 µM) served as positive controls.
Statistical analysis. Data are means ± SE, with differences among results obtained in normoxia versus hypoxia and H/R evaluated by ANOVA or paired Student's t-test as appropriate (18, 24). Significance was accepted for P values < 0.05.
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RESULTS |
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Hypoxia selectively suppresses LPS-induced IL-1 protein
levels in RAW 264.7 cells.
Expression of IL-1
and TNF-
was assessed over 24 h in this murine
monocytic cell line under normoxic conditions and after 1.5 h of
postendotoxemic hypoxia followed by reoxygenation. As seen in Fig.
1, LPS-induced IL-1
protein levels in
cell lysates were suppressed during 1.5 h of exposure to hypoxia but
returned to levels comparable to normoxic control levels within 4 h of reoxygenation. Only trace amounts of IL-1
were secreted into the
medium by any LPS-stimulated RAW 264.7 cells. With respect to
LPS-stimulated TNF-
synthesis, no differences in protein level were
observed in cellular lysates or supernatants by the end of hypoxia or
at any time point measured thereafter compared with the level in
normoxia (Fig. 2). Little IL-1
or
TNF-
protein was detected in unstimulated cells exposed only to DMEM
for 24 h regardless of the presence or absence of subsequent H/R
stresses (data not shown).
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LPS-stimulated steady-state IL-1 RNA levels are
selectively inhibited by hypoxia.
Consistent with the reduced amount of IL-1
protein,
LPS-stimulated IL-1
RNA accumulation quantitated by Northern blot
was significantly suppressed by 1.5 h of hypoxia but returned to
control level within 4 h of reoxygenation of the cells (Fig.
3). In agreement with the data for TNF-
protein, no differences in steady-state TNF-
RNA level were noted
between the normoxic and hypoxic cells at any time point (Fig.
4). Only trace amounts of IL-1
or
TNF-
RNA were detected in cells that were not stimulated with LPS.
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Hypoxia selectively inhibits IL-1 transcription.
Nuclear runoff analyses were performed on LPS-treated RAW 264.7 cells maintained in normoxia throughout or exposed to hypoxic stress,
with the cells assayed at the end of hypoxia (t = 2 h) and
after 1 h of reoxygenation (t = 3 h). As seen in Fig.
5, hypoxia markedly suppressed
IL-1
transcription (~90%), whereas TNF-
transcription at the
same time point was not different from that in normoxic cells. Because
nuclear samples were normalized to equivalent radioactivities, the
transcription of GAPDH was apparently increased slightly by hypoxia,
although mRNA levels for this presumptive housekeeping gene did not
vary by treatment (Figs. 3-5). After 1 h of reoxygenation
(i.e., at t = 3 h), IL-1
transcription had increased to
approximately twice that of normoxic cells at the same time point as
assessed by densitometry, with the results normalized to the GAPDH
signal (data not shown).
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Allo and catalase did not alter hypoxic inhibition of
IL-1.
Intracellular levels of GSH in RAW 264.7 cells showed no evidence of
increased oxidative stress due to LPS or to hypoxia with or without
reoxygenation. By HPLC, the basal [GSH] in cell lysates was
0.62 ± 0.05 nmol/mg total protein. Cell lysate [GSH]
increased to 0.89 ± 0.07 nmol/mg by t = 3 h and then declined
to 0.48 ± 0.05 nmol/mg by t = 24 h regardless of LPS or H/R
exposure. Likewise, we found no differences due to LPS or H/R in
intracellular redox status when assayed by retention of
dichlorofluorescein diacetate, with all cultured RAW 264.7 cells having
comparable emissions at 525 nm when normalized to milligrams of total
protein per well (data not shown). Supernatant lactate dehydrogenase
levels also indicated comparable cell viabilities in the presence and
absence of H/R stress among all LPS-treated cells (data not shown).
Additional experiments were conducted with Allo to exclude a role for
ROS formed via XO, with a concentration that abrogated hypoxic
downregulation of E. coli-induced cytokine expression in
perfused rat livers (23, 30). Here, pretreatment of RAW 264.7 cells
with 50 µM Allo did not prevent the inhibition by hypoxia of
LPS-induced IL-1
protein (Table 1) or
mRNA (data not shown) levels. Likewise, catalase failed to prevent
hypoxia-induced reduction in IL-1
expression (Table
2), again suggesting no significant role
for elevated intracellular ROS in this suppression.
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DISCUSSION |
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We have found that brief hypoxia suppresses LPS-induced expression of
IL-1 but not of TNF-
in RAW 264.7 cells as measured by the
production of protein and specific RNA transcripts. In particular,
IL-1
mRNA expression was reduced by 50-70% 2 and 3 h after LPS
stimulation and was followed by a rapid recovery during reoxygenation
to the control level by 6 h. Differential transcription rates between
hypoxic and normoxic LPS-stimulated cells were also observed for the
IL-1
gene at peak hypoxia (2 h), although not
by 3 h during reoxygenation.
Previous studies suggest that the effects of ambient
PO2 on proinflammatory cytokine gene
expression depend on cell type, the severity of hypoxia and duration of
reoxygenation, and the presence of additional stimuli, notably
bacterial endotoxins. Human peripheral blood-derived macrophages
exposed to PO2 values of 14-147
mmHg without LPS increased IL-1 expression only during normoxic
reoxygenation after at least 6 h of the most severe hypoxia (14 mmHg),
which was dose dependently inhibited by Allo (14). Similarly, THP-1
human macrophage cells cultured under 1 or 9% O2 in
LPS-free medium for up to 24 h secreted suprabasal levels of TNF-
only after at least 4 h at 1% O2 (27). Thus without LPS
stimulation, PO2 values near anoxia
are necessary to affect macrophage cytokine expression. Although the
cells utilized in those reports apparently survived long intervals of
nearly complete anoxia, the clinical relevance of such stress has not been established.
Previous studies in which macrophage-like cells were concurrently
treated with LPS also found significant effects of ambient PO2 on cytokine expression only under
essentially anoxic conditions. In an early report (9), human peripheral
blood monocytes produced more TNF-, IL-1
, and IL-1
in response
to LPS when simultaneously exposed for 24 h to an ambient inspired fraction of O2 of 0%. Likewise, human alveolar macrophages
increased their LPS-induced release of IL-1
when maintained at a
PO2 < 1 mmHg for 24 h (12). Because
these studies evaluated cytokine levels only at 24 h, neither
early-phase hypoxic inhibition of cytokine gene expression nor the
impact of such hypoxic stress on cellular antioxidant defense was
evaluated. Most recently, LPS-treated rat alveolar macrophages exposed
to a PO2 of 13 mmHg for 2 h showed
enhanced release of bioactive TNF-
, although hypoxia without prior
LPS stimulation did not alter mRNA levels for TNF-
, IL-1
, or
IL-1
(19). In contrast to these and all other reports, we found
hypoxic suppression of IL-1
expression after 90 min of exposure to a
PO2 (42 mmHg) resembling that of
venous blood (Figs. 1, 3, and 5) despite unchanged cellular GSH levels.
We also show for the first time that moderate hypoxia that does not
reduce the concentration of critical intracellular antioxidants such as
GSH alters the rate of IL-1
gene transcription (Fig. 5).
These RAW 264.7 cell experiments extend previous studies by Matuschak
et al. (23) and Wibbenmeyer et al. (30) of excised livers in which
E. coli-induced IL-1 and TNF-
expression was inhibited by
secondary H/R. Because of the inherently complex responses by intact
organs such as the liver to live bacteria, these simpler studies may
better identify the basic cellular mechanisms by which hypoxia
modulates proinflammatory cytokine expression. This RAW 264.7 cell
model replicated hypoxia-induced suppression of IL-1
expression seen
in whole livers, but unlike those ex situ liver studies, hypoxia did
not alter TNF-
expression as evaluated here. Furthermore,
pretreatment of RAW 264.7 cells with Allo or catalase did not prevent
hypoxic suppression of IL-1
expression. These differences between
models are not due simply to using purified LPS here rather than live
E. coli because similar results occur in perfused liver with
either stimulus (30; Matuschak GM, Chen Z, and Lechner AJ,
unpublished data).
The molecular mechanisms that underlie this hypoxic inhibition of
IL-1 transcription remain obscure. Loftis et al. (21) recently
reported for perfused rat livers challenged with live E. coli
that a similar period of postbacteremic hypoxia significantly reduced
nuclear translocation of the transcription factor nuclear factor
(NF)-
B from the cytosol. Because transcriptional activation of the
human IL-1
gene depends on NF-
B activation
(13), any interference with this critical event should inhibit IL-1
transcription and lead to reduced IL-1
expression in response to GNB
or LPS. Whether this mechanism plays any role in the inhibition of
IL-1
by hypoxia in RAW 264.7 cells is unknown. Similar to the human IL-1
gene, the murine
IL-1
promoter contains a proximal
NF-
B site (GenBank accession no. U03987), but
its role during endotoxemia has not been proven. The effect of hypoxia
on NF-
B activation in RAW 264.7 cells should be assessed,
particularly because we found disparate results for TNF-
, a cytokine
that contains multiple NF-
B sites in its promoter (3, 6).
Also unlike our results for perfused livers, hypoxic inhibition of
IL-1 expression did not appear to be altered by the redox status of
the cells. Intracellular [GSH] in this study indicated no
increased oxidative stress from either LPS or hypoxia with or without
reoxygenation, with these results supported by cellular retention
levels of dichlorofluorescein diacetate. Our experiments with Allo and
catalase at concentrations that reversed hypoxic suppression in vivo
and in isolated livers (17, 23, 30) suggest no significant role for
XO-derived superoxide or catalase-sensitive hydroxyl radicals in these
RAW 264.7 cell experiments.
Consequently, the disparate results achieved in these studies likely
reflect differences in cell types and the inherently complex
interactions that occur among mixed cell types in intact organs. Based
on well-known differences in LPS responsiveness that exist even among
monocytic cells from the same organism, the signaling pathways and
transcription factors that regulate IL-1 expression may differ
between hepatic Kupffer cells and bloodborne monocytes that are the
likely origin of RAW 264.7 cells. Indeed, hypoxia enhances rather than
suppresses LPS-induced IL-1 and TNF-
synthesis in alveolar
macrophages (12, 18, 19, 25).
Our experiments to determine whether hypoxia-induced suppression of
steady-state IL-1 mRNA depended on de novo protein synthesis were
complicated by the finding that the optimal induction of LPS-induced
mRNA levels was sensitive to CHX even during normoxia. This was an
unanticipated result considering that studies in other monocyte
lineages found that LPS-induced increases in IL-1
mRNA did not
depend on de novo protein synthesis and in which CHX treatment resulted
in superinduction of IL-1
mRNA levels with LPS exposure (8). The
validity of the unexpected results for IL-1
mRNA with CHX was
confirmed by the superinduction of TNF-
mRNA after CHX and LPS in
the same experiments as anticipated (24). Of considerable interest, the
LPS-induced increase of IL-1
mRNA in RAW 264.7 cells was virtually
eliminated by the additive effects of CHX before and hypoxia after LPS
challenge. Thus suppression by hypoxia does not require de novo protein
synthesis because we would argue that if hypoxic suppression of IL-1
transcription was so dependent, then the result for CHX plus LPS plus
hypoxia would have resembled that for CHX plus LPS plus normoxia.
Future studies with this cell line are needed to identify sequences in the IL-1
promoter most sensitive to lowered
PO2.
An important difference between the results in RAW 264.7 cells and our
past buffer-perfused liver studies was the lack of parallel suppression
by hypoxia or reoxygenation of TNF- expression induced by bacterial
substrates. However, this differential responsiveness of IL-1
and
TNF-
to hypoxia is not surprising in light of previous studies.
Beutler (2) and Kruys et al. (15) have shown that regulation of TNF-
expression in macrophages exposed to LPS occurs posttranscriptionally.
Our results in RAW 264.7 cells are in agreement with their findings and
emphasize that, unlike IL-1
, the
TNF-
gene is constitutively active even in quiescent cells.
Adding LPS substantially increased steady-state TNF-
mRNA despite
only a modest increase in transcription rate (Figs. 4 and 5),
indicating that its accumulation is due mainly to posttranscriptional
stabilization. Our results also indicate that hypoxic suppression of
IL-1
is directed at the transcriptional machinery (Fig. 5). We
predict that in any tissue where TNF-
is regulated by
transcriptional activity, hypoxia would downregulate its expression
similar to the effect on IL-1
. It will be important to determine the
mechanisms whereby LPS-induced TNF-
expression is modulated by
ambient PO2 in the intact liver and
ultimately in vivo.
In summary, we have shown that brief hypoxia followed by reoxygenation
differentially regulates proinflammatory cytokine expression in RAW
264.7 cells, with IL-1 but not TNF-
expression reversibly suppressed at the protein, mRNA, and transcriptional levels. The RAW
264.7 cell line allowed us to determine whether such cytokine production is directly modulated by brief periods of H/R in a manner
similar to that for ex situ perfused livers. We found important differences between these systems in that LPS-induced TNF-
synthesis was unaffected by H/R and IL-1
suppression by hypoxia did not appear
to depend on XO-derived superoxide- or catalase-sensitive hydroxyl
radicals. The present results indicate that RAW 264.7 cells can be used
to identify mechanisms whereby hypoxia directly alters cytokine
expression in a homogeneous cell population. Such information will
improve our understanding of the complex role played by cellular redox
status in modulating inflammatory responses in patients who are likely
to develop gram-negative sepsis and shock.
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ACKNOWLEDGEMENTS |
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We thank Cheryl A. Johanns and Drs. Brent Neuschwander-Tetri, James D. Shoemaker, and Zhoumou Chen for critical assistance during the course of these studies.
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FOOTNOTES |
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This work was supported by National Institute of General Medical Sciences Grant R01-GM-43153.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. M. Matuschak, Division of Pulmonary, Critical Care, and Occupational Medicine, Dept. of Internal Medicine, Saint Louis Univ. Hospital, 3635 Vista Ave. at Grand Blvd., St. Louis, MO 63110-0250 (E-mail: matuscgm{at}slu.edu).
Received 29 July 1999; accepted in final form 13 January 2000.
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