Brief hypoxia differentially regulates LPS-induced IL-1beta and TNF-alpha gene transcription in RAW 264.7 cells

Michael M. Ndengele1, Clifford J. Bellone2, Andrew J. Lechner1,3, and George M. Matuschak1,3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
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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)-alpha and interleukin (IL)-1beta 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-alpha and IL-1beta 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-1beta and TNF-alpha concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1beta 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-alpha 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-1beta gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1beta expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1beta expression did not require de novo protein synthesis.

lipopolysaccharide; interleukin-1beta ; tumor necrosis factor-alpha ; septic shock; endotoxin; redox status; cytokines; inflammation; acute respiratory distress syndrome; organ failure; reactive oxygen species


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-1beta and tumor necrosis factor (TNF)-alpha (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-1beta and TNF-alpha 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-alpha , IL-1alpha , and IL-1beta 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-1beta but not of TNF-alpha and IL-1alpha 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-1beta and TNF-alpha gene expression for up to 24 h. As in perfused livers, this brief hypoxia reversibly suppressed IL-1beta 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-1beta expression occurred despite no change in intracellular GSH concentration ([GSH]) due to hypoxia. Surprisingly, LPS-induced increases in TNF-alpha 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-1beta synthesis in RAW 264.7 cells, at least in part by reducing their transcription rate of this canonical proinflammatory cytokine.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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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 at -70°C until thawed in batches for the cytokine assays detailed in Immunoreactive IL-1beta and TNF-alpha analyses. The lysis buffer (300 µl/well) contained 20 mM Tris, pH 7.4, 100 mM NaCl, 1% Triton X-100, and a protease inhibitor mixture consisting of 50 mM NaF, 1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, and 20 µg/ml of aprotinin.

To determine the role of XO-derived ROS in altering LPS-induced cytokine expression during secondary hypoxic stress, parallel experiments were performed in which endotoxin-stimulated cultures were coincubated beginning at t = 0 h with Allo (25, 50, and 100 µM) (30). To ensure complete Allo-induced inhibition of XO, additional studies were also performed in which the cells were preincubated with 50 µM Allo for 18-24 h before the addition of LPS and subsequent analysis of cytokine expression. Similarly, to determine the involvement of hydroxyl ROS in these cytokine expression studies during hypoxia (11, 17, 30), further experiments with bovine catalase (Sigma) were performed. Catalase (100 or 500 U/ml) was added to RAW 264.7 cell cultures either as a pretreatment (30 min before LPS) or as a simultaneous cotreatment with 100 ng/ml of LPS before exposure to hypoxia and analysis as described in Immunoreactive IL-1beta and TNF-alpha analyses. To determine whether de novo protein synthesis was involved in hypoxic modulation of IL-1beta and TNF-alpha gene expression and mRNA turnover, CHX was added to LPS-stimulated cultures (10 µg/ml) before hypoxic exposure and was present until harvest of supernatants and cell lysates for analyses as described in Northern hybridizations.

Immunoreactive IL-1beta and TNF-alpha analyses. Immunoreactive IL-1beta 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-1beta capture antibody and a biotinylated polyclonal anti-murine IL-1beta 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-1beta per milligram of cellular protein. Supernatant and lysate concentrations of immunoreactive TNF-alpha were similarly determined in duplicate with a solid-phase ELISA (17, 25). This assay was sensitive to murine TNF-alpha over a range of 50-3,200 pg/ml with a rabbit anti-murine anti-TNF-alpha 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-alpha and the primary and secondary antibodies were obtained from Genzyme (Cambridge, MA). Interassay coefficients of variation for IL-1beta and TNF-alpha 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-alpha , a 1,301-bp cDNA for murine IL-1beta , 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-1alpha , IL-beta , and TNF-alpha 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.

Nuclei were pelleted by centrifugation (20,000 g at 4°C) and resuspended in 0.225 ml of transcription buffer consisting of 20 mM Tris · HCl (pH, 8.0), 10% glycerol, 6 mM magnesium acetate, 0.3 mM EDTA, 1 mM dithiothreitol, 10 mM NH4Cl, and 84 mM KCl. To this was added 2 mM each ATP, CTP, and GTP, [32P]UTP (250 µCi; Amersham), 10 mM creatine phosphate, and 1 mg/ml of creatine phosphokinase. After incubation of the reaction mixtures (30°C for 25 min), transcripts were isolated by extraction with phenol-chloroform-isoamyl alcohol, chilled at 0°C for 15 min, and precipitated at -20°C with equal volumes of isopropanol. The resulting pellet was resuspended in DNase I buffer (10 mM Tris, pH 7.4, and 10 mM MgCl2) followed by the addition of RNase-free DNase I and incubation (37°C for 15 min). RNA pellets were resuspended in 0.1% SDS, and their radioactivity was determined. For each treatment, counts of 0.5-1.0 × 106 counts/min were hybridized with denatured cDNA and loaded onto baked nylon membranes in 2 ml of hybridization buffer (50% formamide, 6× saline-sodium phosphate-EDTA, 5× Denhardt's solution, 0.1% SDS, and 100 µg/ml of salmon sperm). The cDNA slot blot nylon filters were prehybridized for 1 h, hybridized for 48-72 h at 42°C, and washed, and the filters were exposed to X-ray film for 3-7 days at -70°C, with signals quantitated by densitometry within the linear range. Experiments were performed three separate times, with similar results achieved in each case.

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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ABSTRACT
INTRODUCTION
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Hypoxia selectively suppresses LPS-induced IL-1beta protein levels in RAW 264.7 cells. Expression of IL-1beta and TNF-alpha 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-1beta 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-1beta were secreted into the medium by any LPS-stimulated RAW 264.7 cells. With respect to LPS-stimulated TNF-alpha 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-1beta or TNF-alpha 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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Fig. 1.   Brief hypoxia reduced production of interleukin (IL)-1beta by lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Cell lysates were assayed with an IL-1beta -specific ELISA and for total protein at time points shown after addition of LPS (serotype 055:B5). H/R, hypoxia-reoxygenation. Values are means ± SE of quadruplicate determinations from 5 experiments. Supernatants were found to contain only trace amounts of IL-1beta regardless of treatment or duration of exposure. * Significant difference between hypoxia and normoxia at 2 and 3 h, P < 0.01.



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Fig. 2.   Postendotoxic hypoxia did not alter LPS-induced tumor necrosis factor (TNF)-alpha synthesis in RAW 264.7 cells. Culture supernatants (A) and cell lysates (B) were assayed separately with a specific ELISA at indicated time points after addition of LPS. Values are means ± SE of quadruplicate determinations from 3 experiments. No significant differences were found in secreted or cell-associated TNF-alpha between hypoxic and normoxic treatments.

LPS-stimulated steady-state IL-1beta RNA levels are selectively inhibited by hypoxia. Consistent with the reduced amount of IL-1beta protein, LPS-stimulated IL-1beta 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-alpha protein, no differences in steady-state TNF-alpha RNA level were noted between the normoxic and hypoxic cells at any time point (Fig. 4). Only trace amounts of IL-1beta or TNF-alpha RNA were detected in cells that were not stimulated with LPS.



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Fig. 3.   Steady-state levels of IL-1beta mRNA induced by LPS were reduced by brief hypoxia. Total cellular RNA was loaded (20 µg/lane) and run on agarose gels, blotted, and hybridized with murine 32P-labeled specific cDNA probes (A). +, Presence; -, absence. Densitometry of IL-1beta signals was obtained for triplicate samples and normalized to those for glyceraldehye-3-phosphate dehydrogenase (GAPDH) on corresponding membranes (B). * Significant difference between treatments at 2 and 3 h, P < 0.05.




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Fig. 4.   Hypoxia did not alter increases in steady-state TNF-alpha mRNA due to LPS treatment (100 ng/ml) of RAW 264.7 cells. Northern blot analyses were performed as in Fig. 3, with triplicate TNF-alpha densitometry values normalized to their corresponding GAPDH signals. No differences were found between hypoxic and normoxic treatments at similar time points.

Hypoxia selectively inhibits IL-1beta 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-1beta transcription (~90%), whereas TNF-alpha 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-1beta 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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Fig. 5.   Hypoxia decreased transcription of IL-1beta but not of TNF-alpha in LPS-exposed RAW 264.7 cells. Nuclear runoff analyses were performed with approx 2.5 × 107 cells/time point as described in METHODS, with an equal amount of radioactivity loaded for each sample. LPS levels were 100 ng/ml except at time = 0 h. pBS, pBluescript vector that served as negative control. Data are representative of 3 separate experiments. Note high basal TNF-alpha transcription that, like GAPDH, appears to be increased slightly by hypoxia. Quantitative densitometry indicated a significant reduction at 2 h in IL-1beta transcription due to hypoxia, P < 0.001.

Allo and catalase did not alter hypoxic inhibition of IL-1beta . 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-1beta protein (Table 1) or mRNA (data not shown) levels. Likewise, catalase failed to prevent hypoxia-induced reduction in IL-1beta expression (Table 2), again suggesting no significant role for elevated intracellular ROS in this suppression.

                              
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Table 1.   Inhibition of XO-derived ROS formation does not alter hypoxic suppression of LPS-induced IL-1beta production in RAW 264.7 cells


                              
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Table 2.   Catalase treatment does not alter hypoxic suppression of LPS-induced IL-1beta production in RAW 264.7 cells

To determine whether this hypoxia-induced suppression of IL-1beta depended on de novo protein synthesis, the RAW 264.7 cells were treated with CHX (10 µg/ml) before and then during LPS exposure. Surprisingly, CHX itself inhibited the appearance of IL-1beta mRNA by 60% versus LPS-treated control values. No such reduction by CHX was noted on steady-state TNF-alpha RNA for which the levels were significantly greater than with LPS alone. The combination of CHX and hypoxia virtually eliminated LPS-induced IL-1beta mRNA expression (data not shown). Thus the suppressive effect of hypoxia was not reversed by inhibiting de novo protein synthesis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have found that brief hypoxia suppresses LPS-induced expression of IL-1beta but not of TNF-alpha in RAW 264.7 cells as measured by the production of protein and specific RNA transcripts. In particular, IL-1beta 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-1beta 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-1beta 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-alpha 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-alpha , IL-1alpha , and IL-1beta 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-1beta 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-alpha , although hypoxia without prior LPS stimulation did not alter mRNA levels for TNF-alpha , IL-1alpha , or IL-1beta (19). In contrast to these and all other reports, we found hypoxic suppression of IL-1beta 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-1beta 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-1beta and TNF-alpha 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-1beta expression seen in whole livers, but unlike those ex situ liver studies, hypoxia did not alter TNF-alpha expression as evaluated here. Furthermore, pretreatment of RAW 264.7 cells with Allo or catalase did not prevent hypoxic suppression of IL-1beta 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-1beta 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)-kappa B from the cytosol. Because transcriptional activation of the human IL-1beta gene depends on NF-kappa B activation (13), any interference with this critical event should inhibit IL-1beta transcription and lead to reduced IL-1beta expression in response to GNB or LPS. Whether this mechanism plays any role in the inhibition of IL-1beta by hypoxia in RAW 264.7 cells is unknown. Similar to the human IL-1beta gene, the murine IL-1beta promoter contains a proximal NF-kappa B site (GenBank accession no. U03987), but its role during endotoxemia has not been proven. The effect of hypoxia on NF-kappa B activation in RAW 264.7 cells should be assessed, particularly because we found disparate results for TNF-alpha , a cytokine that contains multiple NF-kappa B sites in its promoter (3, 6).

Also unlike our results for perfused livers, hypoxic inhibition of IL-1beta 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-1beta 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-alpha synthesis in alveolar macrophages (12, 18, 19, 25).

Our experiments to determine whether hypoxia-induced suppression of steady-state IL-1beta 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-1beta mRNA did not depend on de novo protein synthesis and in which CHX treatment resulted in superinduction of IL-1beta mRNA levels with LPS exposure (8). The validity of the unexpected results for IL-1beta mRNA with CHX was confirmed by the superinduction of TNF-alpha mRNA after CHX and LPS in the same experiments as anticipated (24). Of considerable interest, the LPS-induced increase of IL-1beta 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-1beta 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-1beta 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-alpha expression induced by bacterial substrates. However, this differential responsiveness of IL-1beta and TNF-alpha to hypoxia is not surprising in light of previous studies. Beutler (2) and Kruys et al. (15) have shown that regulation of TNF-alpha 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-1beta , the TNF-alpha gene is constitutively active even in quiescent cells. Adding LPS substantially increased steady-state TNF-alpha 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-1beta is directed at the transcriptional machinery (Fig. 5). We predict that in any tissue where TNF-alpha is regulated by transcriptional activity, hypoxia would downregulate its expression similar to the effect on IL-1beta . It will be important to determine the mechanisms whereby LPS-induced TNF-alpha 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-1beta but not TNF-alpha 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-alpha synthesis was unaffected by H/R and IL-1beta 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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