Acute hypoxia increases alveolar macrophage tumor necrosis factor activity and alters NF-kappa B expression

Sandra K. Leeper-Woodford1 and Kristina Detmer2

Departments of 1 Physiology and 2 Biochemistry, Mercer University School of Medicine, Macon, Georgia 31207


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Alterations in alveolar macrophage (AM) function during sepsis-induced hypoxia may influence tumor necrosis factor (TNF) secretion and the progression of acute lung injury. Nuclear factor (NF)-kappa B is thought to regulate the expression of endotoxin [lipopolysaccharide (LPS)]-induced inflammatory cytokines such as TNF, and NF-kappa B may also be influenced by changes in O2 tension. It is thus proposed that acute changes in O2 tension surrounding AMs alter NF-kappa B activation and TNF secretion in these lung cells. AM-derived TNF secretion and NF-kappa B expression were determined after acute hypoxic exposure of isolated Sprague-Dawley rat AMs. Adhered AMs (106/ml) were incubated (37°C at 5% CO2) for 2 h with LPS (Pseudomonas aeruginosa, 1 µg/ml) in normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2). AM-derived TNF activity was measured with a TNF-specific cytotoxicity assay. Electrophoretic mobility shift and supershift assays were used to determine NF-kappa B activation and to identify NF-kappa B isoforms in AM extracts. In addition, mRNAs for selected AM proteins were determined with RNase protection assays. LPS-exposed AMs in hypoxia had higher levels of TNF (P < 0.05) and enhanced expression of NF-kappa B (P < 0.05); the predominant isoforms were p65 and c-Rel. Increased mRNA bands for TNF-alpha , interleukin-1alpha , and interleukin-1beta were also observed in the hypoxic AMs. These results suggest that acute hypoxia in the lung may induce enhanced NF-kappa B activation in AMs, which may result in increased production and release of inflammatory cytokines such as TNF.

transcription factor; nuclear factor-kappa B; oxygen; acute lung injury; cytokines


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WITH ITS LOCATION in the O2-rich environment of lung airways, the alveolar macrophage (AM) depends primarily on energy provided by oxidative phosphorylation for its secretory function in a normoxic environment (28). The relative activities of enzymes used in oxidative phosphorylation and glycolysis in AMs are best suited for environments that are well oxygenated, and acute changes in environmental O2 may perturb AM function (21, 28). In sepsis-induced acute lung injury (ALI), airway edema and atelectasis, which may result in low ventilation-perfusion conditions, could result in reduced O2 tension around AMs in the affected lung areas (16, 19, 30). Because AMs play a central role in cytokine secretion and phagocytosis of bacteria and particles in the lung, low O2-induced alterations in macrophage function during ALI may influence the progression of this lung injury (19-21).

Tumor necrosis factor-alpha (TNF), an important early-phase mediator of ALI, is avidly produced by AMs in response to bacterial endotoxin [lipopolysaccharide (LPS)] (13, 16, 20, 30). Previously, investigators (8, 13, 22) demonstrated that TNF secretion in human mononuclear cells and macrophage cell lines may be increased by exposing these cultured cells to hypoxia for 18-24 h. These authors suggested that hypoxia may be a stimulus that induces macrophages to release higher levels of TNF and also of interleukin (IL)-1 during conditions where O2 tension reaches very low levels. Members of the transcription factor nuclear factor (NF)-kappa B family are thought to be involved in the production of inflammatory cytokines such as TNF in macrophages (1, 18, 26). An initial event in the transcriptional regulation of cytokine production is the dissociation of NF-kappa B proteins from Ikappa B inhibitory proteins (1, 12, 17, 24). This allows NF-kappa B to migrate to the nucleus where it binds to specific promoter sites, initiating activation of cytokine gene transcription (1, 12, 18, 24). Inhibition of NF-kappa B activation and attenuation of cytokine production have been observed after treatment with corticosteroids and antioxidants, but regulation of this transcription factor and the inflammatory cytokines induced by NF-kappa B is a multifaceted area of investigation (1, 10, 12, 17, 18, 23, 24).

Although there is evidence that 24 h of exposure to hypoxia may increase TNF secretion in human mononuclear cells and that hypoxia may activate transcription factors in certain cell lines, the NF-kappa B and secretory activities of AMs during very acute stages of hypoxic exposure have not been defined. We hypothesized that reduction in the O2 tension surrounding AMs acutely induces alterations in NF-kappa B activation and TNF production in these lung cells. We proposed to examine AM-derived TNF production after exposure to hypoxia and to determine whether the nuclear transcription factor NF-kappa B is affected by exposure of AMs to low O2. To test this, TNF activity was monitored in conditioned medium, NF-kappa B activation was determined in cell extracts, and cytokine mRNA was measured in isolated rat AMs exposed to acute hypoxia. Electrophoretic mobility shift assays (EMSAs) and RNase protection analyses were performed on cellular extracts of these AMs to determine the NF-kappa B activation and induction of selected mRNAs in these lung cells after acute alterations in O2 tension.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental protocol with isolated AMs. Pulmonary AMs were collected from the bronchopulmonary lavage fluid of male Sprague-Dawley rats (n = 8) with methods similar to those previously described (20, 21). Before lavage, the rats were exsanguinated after intraperitoneal anesthesia with pentobarbital sodium (50 mg/kg; Abbott, Chicago, IL). The lungs were lavaged 10 times in situ with 10-ml aliquots of Ca2+- and Mg2+-free phosphate-buffered saline (Mediatech, Washington, DC). The cell fractions were washed in phosphate-buffered saline, counted, and suspended (106 cells/ml) in Dulbecco's culture medium (DM; Mediatech) containing 1% penicillin-streptomycin (Mediatech) and 5% fetal bovine serum (Sigma, St. Louis, MO). The cells were allowed to adhere (37°C at 5% CO2) to plastic culture dishes (Falcon, Lincoln Park, NJ) for 18 h, the DM and nonadherent cells were then decanted, and 1 ml of fresh DM was added to the adhered AMs. In certain control experiments, AMs were adhered for only 1 h before the experimental protocols to determine whether adherence time affected NF-kappa B activation. Adherence yielded >95% viable adhered AMs as determined with trypan blue exclusion and nonspecific esterase stain (Sigma) on selected preparations (20). The adhered AMs were incubated (37°C at 5% CO2) in normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2) for 2 h with and without LPS (Pseudomonas aeruginosa, 1 µg/ml; List Laboratories, Campbell, CA) added at time 0. The 2-h time point was selected for these studies because previous experiments performed in this laboratory indicated that LPS-induced TNF activity from AMs was measurable and stable at this time (20). Incubations of AMs in normoxia or hypoxia were performed in modular incubation chambers (21) as detailed in Incubation of AMs in normoxia and hypoxia. At the 2-h time point, the cell-free conditioned DM was removed and the adhered AM preparations were rinsed once with phosphate-buffered saline. With the use of trypan blue exclusion (20) on selected AM preparations, cell viability was determined to be >90% in all experimental conditions examined. The 0-time DM samples, the 2-h conditioned DM samples, and the AM preparations were stored at -70°C until time of analysis. Each experiment was performed on two to eight individual rat AM populations.

Incubation of AMs in normoxia and hypoxia. All isolation and adherence procedures for AMs were done in room air. The culture dishes containing adhered AMs with and without LPS were placed in humidified modular incubation chambers (Flow Laboratories, McLean, VA). The chambers were sealed and flushed with the appropriate gas mixture for 10 min, and the sealed modular chambers were incubated (37°C) for 2 h (21). In all experiments, exposure of the cell preparations to 95% air-5% CO2 is referred to as normoxic, whereas hypoxic exposure refers to the preparations in an environment of 95% N2-5% CO2. The percentages of O2 and CO2 in the modular incubation chambers were tested with Beckman gas analyzers (21). In the chambers incubated in normoxia, the O2 level was 21.0 ± 0.3% and CO2 was 5.0 ± 0.4%; in the hypoxic chambers, O2 was 1.8 ± 0.4% and CO2 was 5.0 ± 0.3%.

Preparation of whole cell extracts. Whole cell extracts were prepared by a modification of the extraction method described by Dent and Latchman (6). The following extraction buffer (0.05 ml) was added to each of the frozen, adhered cell preparations: 20 mM HEPES buffer (pH 7.8), 450 mM NaCl, 0.4 mM EDTA, 0.5 mM dithiothreitol (Sigma), 25% glycerol, 50 µg/ml of antipain, 40 µg/ml of bestatin, 50 µg/ml of chymotrypsin, 10 µg/ml of E64, 0.5 µg/ml of leupeptin, 0.7 µg/ml of pepstatin, 100 µg/ml of phosphoramidon, 1 mg/ml Prefabloc, and 2 µg/ml of aprotinin (Boehringer Mannheim, Indianapolis, IN). The cellular material was scraped into Eppendorf tubes, treated to two more freeze-thaw cycles (thaw at 37°C), and then centrifuged (13,000 g for 10 min; Marathon MicroA). The supernatants were immediately analyzed for the presence of transcription factors by EMSA (6). The protein content of each of the extracts was determined with the Bio-Rad assay kit (Melville, NY), which is based on the Bradford assay (3).

EMSA. Active NF-kappa B isoforms present in the AM extracts were detected with the EMSA (6). The oligonucleotide containing the NF-kappa B consensus binding site was purchased from Promega (Madison, WI). This NF-kappa B oligonucleotide contained the sequence AGTTGAGGGGACTTTCCCAGGCTCAACTCCCCTGAAAGGGTCCG. The NF-kappa B oligonucleotide was end labeled by incubation of the oligonucleotide with [gamma -32P]ATP (3,000 Ci/mmol; Amersham, Arlington Heights, IL) and T4 polynucleotide kinase according to standard protocols (Maniatis Manual). Purification of the labeled oligonucleotide was with the QIAquick Nucleotide Removal kit (Qiagen) according to the manufacturer's instructions. Each 20-µl assay contained 5 µl of the prepared whole cell extract, 10 fmol of the NF-kappa B oligonucleotide end labeled with 32P, 0.25 µg of poly dI-dC (Boehringer Mannheim), 0.5 mM dithiothreitol, 4% glycerol, and 20 mM Tris (pH 7.5). For competition experiments, the assays also contained 1 pmol of unlabeled homologous or heterologous competitor oligonucleotide that was added to the reaction mixture before the addition of labeled oligonucleotide. After a 15-min incubation at room temperature, each of the prepared whole cell extracts was loaded onto a nondenaturing 5% polyacrylamide gel. The gels were run at 200 V for 1 h, 20 min in 0.5× Tris-borate-EDTA (0.045 M Tris borate and 1 mM EDTA). After electrophoresis, the gels were dried and autoradiographed at -70°C with an intensifier screen. Bands corresponding to NF-kappa B were identified by competition experiments: the homologous cold oligonucleotide eliminated NF-kappa B binding, whereas the heterologous one did not. Each EMSA gel contained all four conditions, with extracts from a single rat AM population exposed to each condition on each gel. For determining the densities corresponding to the NF-kappa B species, the broadest band of NF-kappa B was analyzed in each lane. To control for variable band intensity caused by changes in the specific activity of the probe and/or exposure times, the intensity of the NF-kappa B band for the normoxic condition was defined as 1 for each gel.

For supershift assays, NF-kappa B antibodies to the isoforms p50, p52, p65, Rel B, and c-Rel (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the binding reaction mixture and incubated for 20 min at room temperature subsequent to the incubation of labeled oligonucleotide probe with the whole cell extract. The antibodies used were specific to the isoform indicated and specifically reactive to the rat proteins. After incubation, the reaction mixture was electrophoresed as above. The gel was dried and exposed for autoradiography. Autoradiographs were scanned by densitometry, and band densities were quantified as relative densitometry units with the National Institutes of Health (NIH) Image program.

RNase protection assay. RNA was extracted from AMs by the acid guanidinium-phenol-chloroform method of Chomczynski and Sacchi (5). The DNA templates used in generating the cytokine probes were the rCK-1 Multi-Probe Template Set (PharMingen, San Diego, CA) that can be used to specifically target rat mRNAs encoding IL-1alpha , IL-1beta , TNF-alpha , TNF-beta , IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, and interferon (IFN)-gamma as well as L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The probes were generated by transcribing with T7 RNA polymerase in the presence of [alpha -32P]UTP (800 Ci/mmol). Hybridization (55°C for 18 h) and RNase protection were carried out with the RPA II kit (Ambion, Austin, TX). After electrophoresis (200 V for 2 h) through a 7 M urea-5% acrylamide gel, the gel was dried and autoradiographed. Quantification of band densities was determined with the NIH Image program. For each cytokine, the densitometry units under normoxia for each rat were compared with the densitometry units under hypoxia for the same rat, enabling a direct comparison of the increased RNA for each species. RNA from each AM preparation was analyzed by RNase protection four times, giving comparable results in each of the four rat AM populations.

TNF assay. The conditioned DM samples were assayed for TNF activity with the L929 cytotoxicity assay (19, 20). Briefly, L929 mouse fibroblast cells in DM were grown to confluence in flat-bottom 96-well plates, and fresh DM with actinomycin D (5 µg/ml) was added to each well. One hundred microliters of each of the following were added to duplicate wells of L929 cells: DM alone to represent 0% cytotoxicity, AM-conditioned medium samples, and DM over blank wells to represent 100% cytotoxicity. The plates were incubated (37°C at 5% CO2) for 20 h, and the remaining L929 cells were stained for 10 min with 0.5% crystal violet in 20% methanol, rinsed, and air-dried. With a microplate reader, the optical density (550 nm) of the stained cells and the percent L929 cytoxicity were determined. One unit of TNF activity equals 50% L929 cytotoxicity (19, 20).

Statistical analysis. One-way analysis of variance and paired t-tests were used with Tukey's range tests to test for significant differences between groups (29). The level of significance was assigned at P < 0.05 (29).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxia induces increased TNF activity. Compared with the time 0 TNF activity of the AM preparations (0.5 ± 0.1 and 0.3 ± 0.4 U/ml for control and LPS-exposed AMs, respectively), the conditioned medium of control, non-LPS-exposed AMs in normoxia for 2 h showed no significant increase in TNF activity (Fig. 1). The medium of control AMs exposed to hypoxia for 2 h showed a trend for increased TNF activity compared with the activity at time 0 and 2 h in control AMs incubated in normoxia. In AM preparations exposed to either normoxia or hypoxia, TNF activity was significantly increased in conditioned medium of AMs exposed to LPS. Compared with the LPS-induced TNF activity of AM preparations exposed to normoxia for 2 h, the conditioned medium of AMs exposed to LPS and hypoxia for 2 h demonstrated significantly increased TNF activity.


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Fig. 1.   Tumor necrosis factor (TNF) activity of endotoxin [lipopolysaccharide (LPS)]-stimulated alveolar macrophages (AMs) exposed to normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2). AMs were isolated from rats, adhered, and were then incubated with and without LPS (Pseudomonas aeruginosa, 1.0 µg/ml) for 2 h in normoxia or hypoxia. Baseline time 0 TNF activity of AM preparations was 0.5 ± 0.1 and 0.3 ± 0.4 U/ml for control and LPS-exposed AMs, respectively. Values are means ± SE of triplicate assays on conditioned medium of experiments with 106 AMs from each of 8 rats. * P < 0.05 for control vs. LPS in respective gases. ** P < 0.05 for control vs. LPS in respective gases and for LPS exposed in normoxia vs. hypoxia.

Hypoxia induces enhanced NF-kappa B DNA binding. LPS-stimulated AMs demonstrated increased NF-kappa B binding, and the binding was completely inhibited by preincubation of nuclear extracts with an excess of unlabeled consensus oligonucleotide competitor (Fig. 2). The binding was unchanged when cold heterologous competitor activator protein-1 oligonucleotide was included. Compared with the LPS-induced NF-kappa B binding observed in AMs in normoxia, 2 h of hypoxic exposure enhanced the LPS-induced NF-kappa B binding. To confirm the identity of the NF-kappa B isoforms, nuclear extracts from the LPS-exposed AMs were treated with antibodies to the p50, p52, p65, Rel B, and c-Rel protein components of NF-kappa B (Fig. 2B). The nuclear extracts showed shifts with antibodies to p65 and c-Rel, with p65 inducing the most dramatic shift of the NF-kappa B complex. In a series of experiments examining control, non-LPS-exposed AMs incubated for 2 h in normoxia or hypoxia, NF-kappa B binding activity in the AMs exposed to hypoxia alone showed enhanced expression of NF-kappa B binding (Fig. 3).




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Fig. 2.   Enhanced activation of LPS-induced nuclear factor (NF)-kappa B binding in AMs exposed to hypoxia. A: electrophoretic mobility shift assay (EMSA) of nuclear protein extracted from AMs exposed for 2 h to normoxia (lane 1), normoxia with (+) LPS (lanes 2-4), or hypoxia + LPS (lanes 5 and 6). Minimal NF-kappa B binding was observed in normoxic control AMs (lane 1). Hypoxia enhanced LPS-induced NF-kappa B binding (lane 5) compared with LPS-induced binding observed in normoxia (lane 2). In extracts of AMs incubated in normoxia, addition of 100-fold excess unlabeled activator protein (AP)-1 heterologous oligonucleotide (oligo; lane 3) did not affect NF-kappa B binding, but addition of 100-fold excess unlabeled NF-kappa B homologous oligo competed for LPS-induced NF-kappa B binding and eliminated specific band (lane 4). Results are representative of 4 repeated experiments. B: supershift EMSA of nuclear proteins extracted from AMs exposed for 2 h to hypoxia (lanes 3-10). Minimal NF-kappa B binding was observed in normoxic control AMs (lane 1). Compared with AMs exposed to LPS in normoxia (lane 2), LPS-induced NF-kappa B binding was enhanced by hypoxia (lane 3). Addition of antibodies to p65 and c-Rel caused supershifts with a decrease in NF-kappa B binding intensity (lanes 7 and 10). Antibodies to p50, Rel B, and p52 did not result in supershifts (lanes 6, 8, and 9). C: densitometry of EMSA showing enhanced expression of NF-kappa B in AMs exposed to LPS and hypoxia for 2 h. Values are means ± SE of experiments with 106 AMs from each of 4 rats. * P < 0.05 vs. normoxia/control. ** P < 0.05 vs. normoxia/control and normoxia/LPS.




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Fig. 3.   Enhanced activation of NF-kappa B binding in control AMs exposed to hypoxia. A: EMSA of nuclear protein extracts from 2 individual populations of isolated and adhered AMs exposed for 2 h to normoxia (lanes 1 and 2) or hypoxia (lanes 3 and 4). Addition of 100-fold excess of unlabeled homologous oligo competed for NF-kappa B binding and eliminated specific bands induced by hypoxia (lane 5). Results are representative of 4 repeated experiments. B: densitometry of EMSA showing enhanced expression of NF-kappa B in AMs exposed to hypoxia for 2 h. Values are means ± SE of experiments with 106 AMs from each of 4 rats. ** P < 0.05 vs. normoxia/control.

Hypoxia induces enhanced cytokine mRNA. Incubation for 2 h with LPS induced mRNA bands for IL-1beta , TNF-alpha , and IL-1alpha in AMs exposed to normoxia or hypoxia, but control, non-LPS-exposed AMs in either normoxia or hypoxia showed no mRNA bands for these cytokines (Fig. 4A). Compared with the LPS-induced cytokine mRNA of AMs exposed to normoxia for 2 h, AMs exposed to LPS and hypoxia for 2 h demonstrated enhanced mRNA bands for IL-1alpha , IL-1beta , and TNF-alpha (Fig. 4). In addition, compared with the mRNA bands of normoxia-exposed AMs, the bands for IFN-gamma , L32, and GAPDH appeared enhanced in the hypoxia-exposed AMs incubated with and without LPS (Fig. 4A).



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Fig. 4.   Induction of mRNA in AMs exposed for 2 h to LPS in normoxia or hypoxia. A: RNase protection assay showing expression of cytokine and other AM protein mRNAs in total RNA purified from 2 individual AM populations (106 AMs/condition). Hypoxia induced enhanced expression of LPS-induced cytokines interleukin (IL)-1beta , IL-1alpha , and TNF-alpha in AMs. Hypoxia alone induced expression of interferon (IFN)-gamma , L32, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in AMs with or without LPS present. neg, Negative. B: densitometry of RNase protection assay of LPS-exposed AMs incubated in normoxia or hypoxia. Values are means ± SE of experiments with 106 AMs from each of 4 rats. Band densities show enhanced expression of IL-1alpha , IL-1beta , and TNF-alpha in AMs exposed to LPS and hypoxia for 2 h. * P < 0.05 compared with respective normoxia control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of our experiments indicate that compared with AMs exposed to normoxia, AMs exposed for 2 h to hypoxia secrete higher levels of TNF when stimulated in vitro with LPS. In a previous study, Leeper-Woodford and Mills (21) reported that rabbit AMs had reduced ATP levels and decreased retention of phagocytosed particles within the first 30-60 min of hypoxic exposure. Data from this previous study demonstrated that the ATP response to hypoxia is rapid and that AM function may be dramatically altered by low O2. The results of our present studies now indicate that the early-response mechanisms of AMs to hypoxia may also induce altered cytokine secretion in AMs and that very acute hypoxic exposure upregulates LPS-induced TNF release from these lung macrophages. Previously, investigators have suggested that hypoxia upregulates TNF production by macrophages. Kisala et al. (16) noted enhanced TNF and IL-1 production from AMs after pulmonary atelectasis and associated the enhanced fever response to these elevated cytokine levels from AMs in collapsed areas of the lung. These authors suggested that hypoxia was the most likely cause for the changes in AM activation. Ertel et al. (7) demonstrated that 60 min of hypoxemia induced release of TNF and other proinflammatory cytokines in mouse plasma and found that peritoneal macrophages and Kupfer cells from these mice produced increased levels of TNF, IL-1beta , and IL-6 in vitro. Hempel et al. (13) and other investigators (8, 22) have demonstrated that in vitro exposure to 24 h of hypoxia increased the LPS-stimulated release of TNF in human mononuclear cells and a macrophage cell line. Our results not only support these previous observations that hypoxia induces increased TNF release but also provide evidence that hypoxia induces alterations in TNF production and secretion in AMs at very acute time points.

To further characterize the nature of the effect of hypoxia and LPS on TNF production in AMs, we analyzed the effect of acute hypoxic exposure on NF-kappa B activation. In our present studies, NF-kappa B activation was increased by exposure of AMs to acute hypoxia. These results suggest that the stimulatory effects of hypoxia on TNF activity may be mediated in part through increased activation of NF-kappa B, a transcription factor that may represent a central pathway for regulation of the expression of multiple proinflammatory mediators including TNF, IL-1, IL-2, IL-6, IL-8, and certain stress-response proteins. These factors resulting from NF-kappa B activation are responsible for many early responses to bacterial infection or LPS exposure and may play essential roles in conditions such as ALI (1, 18, 26).

NF-kappa B is a heterodimeric complex containing the Rel isoforms p50, p52, p65, Rel B, and c-Rel, and in resting cells, NF-kappa B is sequestered in the cytoplasm bound with the Ikappa B inhibitor (1, 10, 12, 23, 24). With LPS stimulation, Ikappa B is phosphorylated and degraded in the cell cytoplasm so that free NF-kappa B dimers can then translocate to the nucleus and bind with high-affinity sites in the promoter regions of target genes that stimulate transcription of those genes (1, 10, 12, 26). Although studies have indicated that free NF-kappa B binds to DNA as heterodimers of the p50 and p65 subunits to initiate transcription, Schmitz and Baeuerle (24) found that transient expression of p65 alone could also result in gene transcription. In addition, these authors and others (1, 10, 12, 17) have suggested that p50 dimers could act as regulators of NF-kappa B activity and actually suppress the transcriptional activity of p65 subunits. With respect to the subunit composition required for activation of gene transcription, the time course of transcription factor activation may also be crucial (1, 24). It has been proposed that appearance of the p50 isoform may be a later activation or downregulator event and that activation of specific transcription factor isoforms may vary depending on the times examined after stimulation of the cells (1, 24). Dimer composition of the transcription factor isoforms may determine the fine DNA-binding specificity of a given NF-kappa B complex in that varying isoforms may be a way to selectively control transcriptional activation (1).

In the present studies, we found increased p65 and c-Rel isoforms of NF-kappa B in the LPS-stimulated AMs exposed to acute hypoxia, with the p65 isoform of NF-kappa B appearing to be the predominant one in this macrophage system. Hansen et al. (10) have also noted that c-Rel may be an important subunit in NF-kappa B activation in that c-Rel may complex with p65 to initiate transcription. Our results agree with these findings and indicate that in the AM system, the LPS-inducible protein-DNA complexes assembled on the NF-kappa B binding sites contain p65 and possibly c-Rel proteins. Because our studies examined very early LPS- and hypoxia-induced activation of AMs, it is possible that activation of the specific isoforms of NF-kappa B is time dependent and that our findings indicating early activation of p65 and c-Rel may reflect the immediate-response elements of LPS- and hypoxia-induced NF-kappa B activation.

The present studies also demonstrate increased mRNA for TNF, IL-1beta , and IL-1alpha in the AMs exposed to acute hypoxia. Because the TNF, IL-1alpha , and IL-1beta genes contain NF-kappa B binding sites in their promoters (1, 18, 26), the increased intensity of these RNA bands in the LPS-induced, hypoxia-exposed AMs is consistent with the increased NF-kappa B activity detected in these lung cells. These results are also consistent with previous reports (18, 30) that LPS in vivo induces TNF and IL-1 as the earliest responders from the lung in sepsis and supports the proposal that these macrophage cytokines play a central role in the early pathogenesis of LPS- and bacterial-induced injury to the lung.

Additional findings in our studies indicate that mRNA for IFN-gamma , GAPDH, and L32 may be slightly increased in the control and LPS-exposed AMs incubated in hypoxia. It has been reported (27) that IFN-gamma expression may be increased through a TNF-dependent mechanism. It is therefore possible that the enhanced TNF production we observed in AMs exposed to hypoxia may induce IFN-gamma through activation of an IFN-gamma -responsive factor that is also known to have an NF-kappa B site (27). Our observations of slightly enhanced GAPDH and L32 bands for mRNA may be consistent with the stress of hypoxia inducing cellular activation (2, 7, 9, 14, 16, 17, 28). In AMs and other cells, the glycolytic pathways are rapidly induced by a low-O2 environment (9, 28), and under hypoxic conditions, the increased dependence on glycolysis as an energy source is consistent with the increased levels of GAPDH mRNA in our preparations. The ribosomal protein L32 is involved in translation of mRNA in actively synthesizing ribosomes (15). There is evidence that an ATP-dependent protein may be involved in ribosome activation, and it is possible that this protein or low ATP-induced cellular stress may influence L32 mRNA expression during the enhanced synthesis of cytokine or stress proteins in the AMs exposed to hypoxia (9, 15). In addition, it should be noted that mRNA stability may be affected by altered ATP levels in that ATP may be required for mRNA degradation (11). In hypoxic cells with low ATP, it may be that reduced mRNA degradation occurs that allows for increased or prolonged expression of mRNA for certain cellular proteins (11).

Although Hempel et al. (13) proposed that hypoxia-induced production of TNF and IL-1 was due to decreased PGE2 synthesis during 24 h of hypoxic exposure in macrophages, the mechanism for acute hypoxia-mediated increased TNF production could possibly involve other factors such as reactive oxygen species that could behave like second messengers to activate transcription involving NF-kappa B (2, 4, 9, 14, 17, 23-25, 27). In addition, there is evidence that phosphorylation events, which may be involved in regulating transcription, may be altered by hypoxia (17). An interesting study by Koong et al. (17) demonstrated that hypoxia caused activation of NF-kappa B by inducing tyrosine phosphorylation of Ikappa B, an important proximal step that precedes its dissociation from the NF-kappa B cytoplasmic complex before transcriptional activation. Although no conclusions can be made as to the mechanism of NF-kappa B activation and increased TNF production and release in acute hypoxia, the dramatic changes in oxidative metabolism and reduction in ATP levels in AMs during acute exposure to low environmental O2 may undoubtedly lead to complex alterations in transcription, translation, and secretion in these lung cells.

The present studies demonstrate that acute hypoxic exposure may play a role in the regulation of inflammatory genes through upregulation of NF-kappa B, a transcription factor critical to the activation of cytokine genes that may be induced in acute inflammation. Our results also provide evidence that hypoxia may be a potent regulatory mechanism of lung cell activation during exposure to LPS and other acute inflammatory states. Hypoxia may occur during ALI in patients with sepsis, and increased cytokine production has been implicated as a primary inducer of many facets of ALI. Attenuation of the activation of NF-kappa B may be of great benefit in altering the acute cytokine responses that are involved in ALI. Because NF-kappa B activation leads to enhanced expression of many inflammatory cytokines, modulation of NF-kappa B activation may provide a direct method of inhibiting proinflammatory mediators. Manipulation of transcription factors such as NF-kappa B may be useful for altering biological processes such as the cytokine cascade that may induce ALI, and evaluation of potential modulators of NF-kappa B activation is needed. Our results provide further insight into the role of acute hypoxia in lung cell activation, and these studies may lead to potential targets for intervention during disease states such as sepsis-induced ALI.


    ACKNOWLEDGEMENTS

S. K. Leeper-Woodford was supported in part by National Heart, Lung, and Blood Institute Grant HL-52917 and an American Lung Association of Georgia Research Grant.


    FOOTNOTES

Address for reprint requests and other correspondence: S. K. Leeper-Woodford, Dept. of Physiology, Mercer Univ. School of Medicine, Macon, GA 31207 (E-mail: leeper.sk{at}gain.mercer.edu).

Received 10 September 1997; accepted in final form 22 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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5.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

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