Hypothermia prolongs activation of NF-{kappa}B and augments generation of inflammatory cytokines

Karen D. Fairchild,1 Ishwar S. Singh,2 Sandip Patel,1 Beth E. Drysdale,4 Rose M. Viscardi,1 Lisa Hester,2,3 Heather M. Lazusky,1 and Jeffrey D. Hasday2,3,4

Departments of 1Pediatrics and 2Medicine, University of Maryland School of Medicine; 3University of Maryland at Baltimore Cytokine Core Laboratory; and 4Medicine and Research Services of the Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201

Submitted 17 November 2003 ; accepted in final form 1 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
While moderate hypothermia is protective against ischemic cardiac and brain injury, it is associated with much higher mortality in patients with sepsis. We previously showed that in vitro exposure to moderate hypothermia (32°C) delays the induction and prolongs the duration of TNF-{alpha} and IL-1{beta} secretion by lipopolysaccharide (LPS)-stimulated human mononuclear phagocytes. In the present study, we extended these observations by showing that moderate hypothermia exerts effects on TNF-{alpha} and IL-1{beta} generation in the human THP-1 monocyte cell line that are similar to those that we previously found in primary cultured monocytes; that hypothermia causes comparable changes in cytokine generation stimulated by zymosan, toxic shock syndrome toxin-1, and LPS; and that hypothermia causes similar changes in TNF-{alpha} and IL-1{beta} mRNA accumulation. TNF-{alpha} mRNA half-life, determined after transcriptional arrest with actinomycin D, was not significantly prolonged by lowering incubation temperature from 37 to 32°C, suggesting that hypothermia modifies TNF-{alpha} gene transcription. This finding was further supported by reporter gene studies showing a threefold increase in activity of the human TNF-{alpha} promoter at 32 vs. 37°C. Electrophoretic mobility shift assay revealed that hypothermia prolonged NF-{kappa}B activation, identifying a potential role for this transcription factor in mediating the effects of hypothermia on TNF-{alpha} and IL-1{beta} production. Delayed reexpression of the inhibitor I{kappa}B{alpha}, shown by Northern blotting and immunoblotting, may account in part for the prolonged NF-{kappa}B activation at 32°C. Augmentation of NF-{kappa}B-dependent gene expression during prolonged exposure to hypothermia may be a common mechanism leading to increased lethality in sepsis, late-onset systemic inflammatory response syndrome after accidental hypothermia, and neuroprotection after ischemia.

tumor necrosis factor; I{kappa}B; monocyte; lipopolysaccharide; interleukin-1


THE CYTOKINES TNF-{alpha} AND IL-1{beta} are critical mediators in the host response to infection and injury. While both are essential for optimal host defense against microbial pathogens (18, 19), these cytokines also can induce host tissue injury, shock, and death (11, 65, 67). Their potential for lethality depends, in part, on the combination in which they are expressed. For example, coexposure to TNF-{alpha} and IL-1{beta} (67) or to TNF-{alpha} and interferon (IFN)-{gamma} (21) increases the lethality of these cytokines. Prolongation of cytokine expression increases the likelihood of exposure to a potentially lethal cytokine combination. The dangers of dysregulated cytokine expression have led to stringent and redundant regulation of transcription (38, 55), mRNA stability (26, 38, 44), and translational efficiency (10, 31). Transcription of both cytokines is activated by the proinflammatory transcriptional activator NF-{kappa}B (22).

Engagement of Toll-like receptor (TLR)-4 on macrophages by bacterial lipopolysaccharide (LPS) triggers recruitment of the adapter molecule MyD88, IL-1 receptor-associated kinase, and TNF receptor-associated factor 6 to the receptor. Assembly of this complex activates a protein kinase cascade that leads to phosphorylation of the NF-{kappa}B inhibitor proteins I{kappa}B{alpha} and I{kappa}B{beta}. Phosphorylation of I{kappa}B{alpha} and I{kappa}B{beta} targets them for ubiquitination and proteasomal degradation, thereby permitting active NF-{kappa}B to translocate to the nucleus and activate transcription (4). Reexpression of I{kappa}B{alpha}, which is itself transcriptionally activated by NF-{kappa}B (54), limits ongoing NF-{kappa}B-dependent transcription by facilitating export of NF-{kappa}B (1) from the nucleus to the cytoplasm via the CRM-1-dependent nuclear export pathway (63).

Body temperature is a critical interactive element of the immunologic response to infection (32). The same cytokines that activate the acute phase response and initiate early immunologic defenses, TNF-{alpha}, IL-1{beta}, IL-6, and IFN-{gamma}, also stimulate a transient increase in core temperature during infection (20). Exposure to febrile temperatures enhances innate immune defenses (9, 36) and modifies patterns of cytokine expression (27, 28, 36, 37), and the presence of fever during infection is associated with improved outcomes in humans (32) and animals (2, 9, 17, 36, 56). In contrast to fever, most endothermic animals vigorously defend against hypothermia. In healthy adult humans, neuroendocrine and vasomotor mechanisms act together to maintain core body temperature within a narrow range (36.6–37.6°C). Subcutaneous thermal insulation provides for a steep temperature gradient from the cutaneous surface to the core so that during limited environmental cold exposure, whereas mean body temperature may drop by as much as 5°C, core temperature rarely decreases by more than 1.5°C (68). However, core temperature less than 35°C is a frequent occurrence in neonates and the elderly, whose thermoregulatory mechanisms are impaired, and in patients with pathological conditions such as trauma, prolonged environmental cold exposure, and sepsis. In trauma victims, mortality is inversely correlated with body temperature (39, 46), and mortality is also high in patients with accidental hypothermia, in whom death often results from a late complication rather than from acute effects of hypothermia (12). Furthermore, hypothermia during sepsis is associated with increased mortality and, in some patients, higher circulating levels of TNF-{alpha} and IL-6 (3).

We previously reported that exposing human macrophages to moderate hypothermia (32°C) in vitro modified the kinetics of bacterial endotoxin (LPS)-stimulated TNF-{alpha} and IL-1{beta} secretion (28). In that study, the onset of TNF-{alpha} and IL-1{beta} secretion was modestly delayed, but the duration of cytokine generation was increased so that the level of each cytokine 24 h after stimulation with LPS was more than 50% higher in the 32°C cultures than in the 37°C cultures. In the present study, we used the THP-1 human promonocyte cell line to analyze the mechanisms by which exposure to moderate hypothermia modifies LPS-stimulated TNF-{alpha} and IL-1{beta} expression. We found that the onset of TNF-{alpha} and IL-1{beta} mRNA accumulation and cytokine secretion is delayed but that once initiated, the duration of TNF-{alpha} and IL-1{beta} protein and mRNA expression is prolonged in 32°C THP-1 cell cultures compared with 37°C THP-1 cultures. Reporter gene studies provided further evidence that hypothermia enhances TNF-{alpha} gene transcription. Electrophoretic mobility shift assay (EMSA) showed that hypothermia prolongs activation of NF-{kappa}B, and this change is paralleled by delayed reexpression of the inhibitory I{kappa}B{alpha}. We suggest that hypothermia during infection might cause a dysregulated and counterproductive host immune response, in part by causing critical changes in the kinetics of NF-{kappa}B activation and inactivation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. THP-1 cells obtained from American Type Culture Collection (Manassas, VA) were cultured in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.5 x 10–6 M 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT). Cells were cultured in a 37°C incubator in 5% CO2-enriched air and used for experiments between passages 12 and 20. Vitamin D3 (Biomol, Plymouth Meeting, PA) was added at 10–8 M for 3–4 days to induce differentiation (58).

ELISA for TNF-{alpha} and IL-1{beta}. THP-1 cells were plated at a density of 1 x 106 cells/ml in 48-well plates and preincubated for 30 min in 32 or 37°C incubators. Temperatures were verified by direct measurement with calibrated thermometers. LPS (Escherichia coli 055:B5; Sigma, St. Louis, MO) was added at 100 ng/ml, and plates were immediately replaced at the designated temperatures. Alternately, cells were stimulated with toxic shock syndrome toxin-1 (TSST-1; Sigma) at 25 ng/ml or with zymosan particles (Zymed, San Francisco, CA) opsonized with heat-inactivated human A-negative serum. In the TSST-1 and opsonized zymosan experiments, polymyxin B (Sigma) was added at 5 µg/ml to block potential activation by contaminating LPS that may have been present in the reagents. In addition, endotoxin testing by Associates of Cape Cod (Falmouth, MA) revealed that there was less than 0.02 U/ml of endotoxin activity in the TSST-1 and less than 0.01 EU/ml endotoxin activity in the opsonized zymosan preparations, and polymyxin B suppressed TNF-{alpha} production in cells treated with a comparable amount of LPS.

Supernatants were collected at various time points and stored at –80°C until ELISA was performed in batches. TNF-{alpha} and IL-1{beta} levels were measured in the University of Maryland at Baltimore Cytokine Core Laboratory by performing two-antibody ELISA with biotin-streptavidin-peroxidase detection with the use of commercially available antibodies and substrates (Endogen, Boston, MA) as previously described (28). Briefly, polystyrene plates (Nunc Maxisorb; Nalge Nunc, Rochester, NY) were coated with capture antibody and blocked with 4% BSA. After being washed, samples or standards diluted in assay buffer were added to the wells and incubated at 37°C for 2 h. Plates were washed, and biotin-conjugated detection antibodies diluted in assay buffer were added. After 1-h incubation at 25°C and washing, streptavidin peroxidase (RDI, Flanders, NJ) was added. The plates were washed, and 100 µl of commercially prepared substrate (TMB; Neogen, Lexington, KY) were added. The A450 (minus A650) was measured with a microplate reader (Molecular Devices, Sunnyvale, CA). A curve was fit to the standards with a computer program (Softmax; Molecular Devices), and cytokine concentrations from each sample were calculated from the standard curve. Samples were analyzed in duplicate. The lower limits of detection were 15 and 3 pg/ml for TNF-{alpha} and IL-1{beta}, respectively.

RNA isolation and Northern blot analysis. Ten million vitamin D3-differentiated THP-1 cells per T-75 flask were prewarmed to 32 or 37°C and then stimulated with 100 ng/ml LPS at the same temperatures. At various time points, cells were collected and total RNA was isolated with a modification of the guanidinium isothiocyanate method of Chomczynski and Sacchi (15). Ten micrograms per sample of RNA were separated on an agarose-formaldehyde gel and transferred to a nylon membrane by capillary action, followed by UV cross-linking. Blots were prehybridized at 42°C for 30 min as previously described (60) before radiolabeled probe was added. Plasmids containing cDNA for human TNF-{alpha}, IL-1{beta}, and I{kappa}B{alpha} were obtained from S. A. Nedospasov (National Cancer Institute Center for Cancer Research, Frederick, MD), D. Carter (Upjohn, Kalamazoo, MI), and S. Ghosh (Section of Immunobiology, Yale University School of Medicine, New Haven, CT), respectively. cDNA fragments were excised, gel purified, and labeled with [{alpha}-32P]dCTP with a random primer labeling kit (GIBCO-BRL). After hybridization for 18 h at 42°C, blots were washed and analyzed by autoradiography, followed by densitometry (Molecular Dynamics, Sunnyvale, CA). In addition, the Northern blots were subjected to PhosphorImager analysis (Molecular Dynamics) of TNF-{alpha}- and IL-1{beta}-specific bands. Equivalent RNA loading was documented by UV visualization and photographic documentation of ethidium bromide-stained 18S and 28S rRNA bands. Blots were stripped and reprobed up to three times.

Analysis of TNF-{alpha} mRNA stability. Vitamin D3-differentiated THP-1 cells were stimulated with 100 ng/ml LPS for 30 min at 37°C, after which prewarmed 32 or 37°C medium containing actinomycin D was added (10 µg/ml final concentration of actinomycin D). At time points from 15 min to 4 h after actinomycin D addition, cells were collected and Northern blot analysis was performed for TNF-{alpha} as described. Membranes were analyzed by phosphorimaging. Data were fit to a first-order decay curve (DeltaGraph; RockWare, Golden, CO), and the TNF-{alpha} mRNA half-life was calculated with the decay constant.

Plasmids, transfection, and reporter gene analysis. A sequence of the human TNF-{alpha} gene (–1,455 to +126 bp) was amplified by PCR with genomic THP-1 cell DNA as the template and the primer pairs 5'-GGTACCCTTACGCGTGCTAGCTAATAGAAGAACATCCAAGGA-3' and 5'-TACCGGAATGCCAAGCTTACAGATCTAAGAGAACCTGCCTGGCAGCTT-3', containing an NheI site and an HindIII site, respectively. Genomic DNA was isolated from THP-1 cells by alkaline lysis and spin column purification (QIAmp DNA mini kit; Qiagen, Valencia, CA). PCR mixtures contained 45 µl of Platinum Taq Supermix (PerkinElmer, Wellesley, MA), 200 ng of DNA, and 20 µM of each primer. After initial 4-min denaturation at 94°C, the reaction mixture was subjected to 30 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 1.5 min, followed by a 7-min final extension. The PCR product was gel purified (QiaQuick; Qiagen), digested with NheI and HindIII, and directionally cloned into the NheI/HindIII sites of the firefly luciferase plasmid pGL3 (Promega, Madison, WI). The sequence was verified by dideoxy terminator sequencing and compared with the published sequence (GenBank accession no. M16441). The control Renilla luciferase reporter plasmid driven by the TK promoter (phRL) was purchased from Promega. The I{kappa}B{alpha} superrepressor was obtained from D. Ballard (Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN) (14).

THP-1 cells differentiated with 10–8 M dihydroxyvitamin D for 24 h were transfected with FuGene 6 (Roche Molecular Biochemicals, Indianapolis, IN). Firefly luciferase test plasmid (2–4 µg) and 0.2–0.4 µg of control Renilla luciferase plasmid were mixed with 15 µl of FuGene 6 in 100 µl of complete medium. In each experiment, sham-transfected cells without plasmid DNA added were prepared as control for autoluminescence at both temperatures. After incubation for 30 min, the FuGene 6-DNA mixture was added to cells in 60-mm dishes. The cells were incubated for an additional 48 h, preincubated at 32 or 37°C for 30 min, and stimulated with 100 ng/ml LPS. Cell lysates were collected at various time points in passive lysis buffer (Promega). Reporter gene expression was analyzed with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. The ratio of firefly to Renilla luciferase was calculated for each condition after subtracting background luminescence from the no-DNA transfection controls. In preliminary experiments, THP-1 cells were cotransfected with the firefly and Renilla luciferase plasmids pGL3 and phRL, each driven by the TK promoter, to verify that the firefly-to-Renilla luciferase activity ratios were comparable at 32 and 37°C at each time point.

EMSA for activated NF-{kappa}B and AP-1. Cell extracts from THP-1 cells stimulated with LPS and cultured at 32 or 37°C for various times were prepared according to the method of Schreiber et al. (57). Cells were lysed in buffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, and 1 mM DTT) and protease inhibitors (Complete Mini; Roche Molecular Biochemicals), and total protein concentration was measured with a commercial reagent on the basis of the Bradford reaction (Bio-Rad Laboratories, Hercules, CA) with a bovine serum albumin standard curve. EMSA was performed on whole cell lysates as previously described (48, 60). Double-stranded oligonucleotide probes for NF-{kappa}B (5'-AGT TGA ATG ACT CAG CCG GAA-3') and AP-1 (5'-GCG TTG ATG ACT CAG CCG GAA-3') were purchased from Promega and Santa Cruz Biotechnology (Santa Cruz, CA), respectively, and end-labeled with [{gamma}-32P]ATP and polynucleotide kinase (Promega). Cell extracts and probes were incubated for 30 min at room temperature in buffer containing 10 mM Tris, pH 7.5, 1 mM DTT, 1 mM EDTA, 10% glycerol, 50 mM NaCl, and 1 µg poly(dI-dC). In supershift experiments, antibodies to c-rel, p50, and p65 (Santa Cruz Biotechnology) were added 30 min before radiolabeled probe was added. Samples were separated on 4% polyacrylamide gels, and, after drying, the gels were exposed to X-ray film. Bands were quantified by laser densitometry (Molecular Dynamics).

Western blot analysis. After 30-min preincubation at 32 or 37°C, THP-1 cells were stimulated with 100 ng/ml LPS and cultured at the respective temperatures. Cell lysates were prepared in buffer C, and samples containing 15 µg/lane total protein were loaded and separated on 10% SDS-polyacrylamide gels, and then samples were electrostatically transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). After blocking with 5% nonfat milk overnight, membranes were probed for 2 h with a 1:5,000 dilution of polyclonal rabbit anti-human I{kappa}B{alpha} or I{kappa}B{beta} (Santa Cruz Biotechnology). Bands were detected with a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) followed by chemiluminescence detection (Renaissance; NEN Life Science Products, Boston, MA). Band intensity was quantified by laser densitometry (Molecular Dynamics) and expressed relative to density at 37°C, time 0 (i.e., before addition of LPS).

Statistics. Data are expressed as means ± SE unless otherwise indicated. Differences between 32 and 37°C at multiple time points were analyzed by performing ANOVA followed by the Student-Newman-Keuls multiple comparison test with SigmaStat for Windows software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypothermia delayed and prolonged TNF-{alpha} and IL-1{beta} secretion. In preliminary experiments, THP-1 cells that were differentiated by 72-h treatment with 10–8 M vitamin D3 generated three- to fivefold more TNF-{alpha} and IL-1{beta} in response to stimulation with LPS than did undifferentiated THP-1 cells. However, the kinetics of cytokine secretion and response to hypothermia were similar in both THP-1 phenotypes (data not shown). All subsequent experiments were performed in vitamin D3-differentiated THP-1 cell cultures.

Before LPS stimulation, little or no TNF-{alpha} or IL-1{beta} was detectable in culture supernatants of THP-1 cells. After the addition of 100 ng/ml LPS, TNF-{alpha} and IL-1{beta} levels rose rapidly in the 37°C culture supernatants, peaking at 2 and 6 h, respectively (Fig. 1). In the 32°C cultures, the peak level of TNF-{alpha} and IL-1{beta} secretion was delayed and cytokine generation was more sustained. Peak TNF-{alpha} and IL-1{beta} levels were 1.6- and 2.2-fold higher in the 32°C cell cultures than in the 37°C cultures. After 24-h stimulation with LPS, the concentrations of TNF-{alpha} and IL-1{beta} in the 32°C culture supernatants were 4.8- and 2.5-fold higher, respectively, than the corresponding 37°C culture supernatants.



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Fig. 1. Effect of moderate hypothermia on lipopolysaccharide (LPS)-stimulated TNF-{alpha} and IL-1{beta} protein production by THP-1 cells. Vitamin D3-differentiated THP-1 cells were preincubated for 30 min at 32 or 37°C before addition of 100 ng/ml LPS. Culture was continued at the designated temperatures, and supernatants assayed for TNF-{alpha} (A) and IL-1{beta} (B) by ELISA at the time points indicated. Data are means ± SE of 3 experiments. *P < 0.05, 32 vs. 37°C.

 
To determine whether the effects of hypothermia were specific to LPS-stimulated cytokine production, we also analyzed the effect of hypothermia on cytokine generation induced by two other structurally and functionally dissimilar macrophage activators (Fig. 2): 1) serum-opsonized zymosan, which activates macrophages through CR3 (CD11b/CD18) (23), TLR2 and TLR6 receptors (51), and dectin-1 (13); and 2) TSST-1, which is thought to activate macrophages after binding to nonpolymorphic regions of major histocompatibility complex molecules (42). Zymosan-induced TNF-{alpha} and IL-1{beta} secretion was similar in magnitude to LPS-induced cytokine secretion, whereas peak cytokine levels in TSST-1-activated cells reached only 5% of these levels. However, culturing THP-1 cells at 32°C caused similar modifications in the magnitude and kinetics of cytokine generation in THP-1 cells treated with each of the three agents. In each case, the cytokine concentrations peaked at higher levels in cell culture supernatants at 32°C than at 37°C. To ensure that cell stimulation by these reagents was not caused by contaminating endotoxin, we added 5 µg/ml polymixin B with TSST-1 and zymosan. In addition, we measured the amount of endotoxin activity in the TSST-1 and zymosan preparations and confirmed that polymyxin B was able to suppress a comparable amount of LPS endotoxin activity in these cells. Importantly, the stability of TNF-{alpha} and IL-1{beta} protein, as determined by sequentially measuring the concentration of recombinant cytokines in cell-free culture medium, was not significantly different at 32 and 37°C. Moreover, THP-1 cell viability, as measured by Trypan blue dye exclusion, neutral red uptake, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) reduction, a marker of mitochondrial function, was comparable after 24-h incubation at 32 and 37°C (data not shown).



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Fig. 2. Effect of hypothermia on TNF-{alpha} production induced by other agonists. THP-1 cells were preincubated at 32 or 37°C and then stimulated with serum-opsonized zymosan (0.5 mg/ml) (A and B) or toxic shock syndrome toxin 1 (TSST-1) (25 ng/ml) (C and D) at the respective temperatures. Polymyxin B (5 µg/ml) was added to TSST-1 and zymosan cultures to control for small amounts of extraneous endotoxin (TSST-1- and zymosan-treated cultures contained <0.02 U/ml endotoxin as measured by limulus amebocyte lysate assay). Supernatants were collected at indicated time points, and ELISA was performed in batches for TNF-{alpha} (A and C) and IL-1{beta} (B and D). Data are means ± SE of 3 experiments. *P < 0.05, 32 vs. 37°C.

 
Accumulation of TNF-{alpha} and IL-1{beta} mRNA was delayed and prolonged at 32°C. To begin to identify the mechanisms by which TNF-{alpha} and IL-1{beta} secretion are modified by hypothermia, we performed Northern blotting to analyze TNF-{alpha} and IL-1{beta} mRNA accumulation in LPS-stimulated THP-1 cells cultured at 32 or 37°C. For both TNF-{alpha} and IL-1{beta}, hypothermia induced changes in mRNA accumulation that paralleled changes in cytokine secretion (Fig. 3). In 37°C cell cultures, TNF-{alpha} mRNA levels peaked 1 h after LPS stimulation, decreased to 20% of peak levels by 6 h, and were undetectable by 24 h. In comparison, in 32°C cell cultures, TNF-{alpha} mRNA levels peaked 2 h after LPS stimulation, remained threefold higher than those of 37°C cell cultures at 6 h after LPS stimulation, and returned to baseline by 24 h. In the same cells, IL-1{beta} mRNA levels also peaked later in cells at 32°C than at 37°C (2–4 h vs. 1 h after addition of LPS). By 24 h after stimulation with LPS, IL-1{beta} mRNA levels returned to baseline in the 37°C cells but remained 4.5-fold higher than baseline in the 32°C cells.



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Fig. 3. Northern blot analysis of TNF-{alpha} and IL-1{beta} mRNA expression at 32 vs. 37°C. After preincubation for 30 min at 32 or 37°C, THP-1 cells were treated with 100 ng/ml LPS and cultured at the respective temperatures. Total RNA was prepared at the indicated time points, and Northern blotting was performed for TNF-{alpha} and IL-1{beta} mRNA. Equivalent RNA loading was verified by UV visualization of ethidium bromide-stained 18S and 28S rRNA bands (bottom rows). The blots shown are representative of 3 separate experiments with similar results.

 
TNF-{alpha} mRNA stability was not significantly altered by hypothermia. The net accumulation of TNF-{alpha} and IL-1{beta} mRNA is determined by rates of transcript synthesis and degradation. Although expression of certain cytokines is regulated posttranscriptionally through modification of transcript stability (30, 34), the stability of TNF-{alpha} mRNA appears to be affected only modestly by macrophage activation (52, 62). To determine whether the changes in cytokine mRNA accumulation that occur in hypothermic THP-1 cell cultures are caused, in part, by transcript stabilization, we analyzed the stability of TNF-{alpha} mRNA in 37 and 32°C THP-1 cultures by measuring the decay in transcript levels after treatment with the transcriptional inhibitor actinomycin D. Cells were treated with 100 ng/ml LPS for 30 min at 37°C, then treated with 10 µg/ml actinomycin D, either switched to 32°C or continued at 37°C incubation temperature, and sequentially analyzed for TNF-{alpha} mRNA levels by Northern blotting (Fig. 4). The half-life of TNF-{alpha} mRNA did not differ significantly at 32°C, suggesting that TNF-{alpha} transcription is augmented in the 32°C cells. We found, as did Godambe et al. (29), that the apparent half-life of IL-1{beta} mRNA in 32 and 37°C THP-1 cells, as measured in the actinomycin D-treated cells, was >8 h as a result of an apparent stabilizing effect of actinomycin D on IL-1{beta} mRNA (data not shown).



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Fig. 4. Effect of hypothermia on TNF-{alpha} mRNA stability. THP-1 cells were stimulated with 100 ng/ml LPS for 30 min at 37°C. Actinomycin D (final concentration of 10 µg/ml) was added in medium prewarmed to 32 or 37°C, and incubation was continued at the respective temperatures. Cells were harvested at the indicated time points, and TNF-{alpha} mRNA expression was analyzed by Northern blotting. Data from 3 separate experiments were fit to a first-order decay equation, and half-life of TNF-{alpha} mRNA was calculated with the decay constant. Half-life of TNF-{alpha} mRNA was 16.4 min at 37°C and 19.5 min at 32°C.

 
Hypothermia enhanced TNF-{alpha} gene transcription. In the 32°C THP-1 cell cultures, the persistence of steady-state TNF-{alpha} mRNA accumulation in the setting of insignificant transcript stabilization suggests that hypothermia prolongs TNF-{alpha} gene transcription. To further prove that TNF-{alpha} transcription is modified in hypothermic cell cultures, we analyzed the effects of hypothermia on the activity of a firefly luciferase reporter plasmid driven by a 1.1-kb fragment of the human TNF-{alpha} 5' flanking sequence and 5' untranslated region (Fig. 5). THP-1 cells were transiently cotransfected with the firefly reporter plasmid and a Renilla luciferase control plasmid and studied 48 h later. To maintain a differentiation state similar to that of the cells used in earlier studies, THP-1 cells were continuously exposed to 10–8 M vitamin D3 for 72 h beginning 24 h before transfection. Transfectants were preincubated at either 32 or 37°C for 30 min, stimulated with 100 ng/ml LPS, and sequentially analyzed for promoter activity. As anticipated on the basis of the previous analysis of steady-state TNF-{alpha} mRNA levels and TNF-{alpha} mRNA stability, TNF-{alpha} transcription was both enhanced and prolonged in the 32°C cell cultures. Of note, we controlled for nonspecific effects of hypothermia on luciferase activity by showing that in cells cotransfected with firefly luciferase and Renilla luciferase plasmids, both driven by the TK promoter, firefly luciferase-to-Renilla luciferase luminescence ratios were comparable at 32 and 37°C at all time points studied.



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Fig. 5. Hypothermia enhances TNF-{alpha} promoter activity. THP-1 cells were transiently cotransfected with a reporter plasmid containing 1.1 kb of the human TNF-{alpha} promoter preceding the firefly luciferase gene and with a control Renilla luciferase plasmid. Transfected cells were stimulated with LPS and cultured at 32 or 37°C. At various time points after addition of LPS, cells were lysed and analyzed by luminometry, and firefly luciferase-to-Renilla luciferase ratios were calculated after subtraction of background luminescence from sham-transfected cells (24 h data shown). In separate experiments, the I{kappa}B{alpha} superrepressor plasmid (I{kappa}{beta}-SR) was cotransfected with the human TNF-{alpha} promoter-luciferase reporter, resulting in 88 and 87% reduction in luciferase activity in 32 and 37°C cell cultures, respectively. In preliminary experiments with plasmids in which firefly luciferase expression and Renilla luciferase expression were driven by the same non-NF-{kappa}B-dependent thymidine kinase promoter, the ratio of firefly luciferase expression to Renilla luciferase expression did not differ at 32 and 37°C for the time course studied (data not shown). Data are expressed as means ± SE of 3 experiments. *P < 0.05, 32 vs. 37°C.

 
Because TNF-{alpha} transcription is in part dependent on the proinflammatory transcription factor NF-{kappa}B, we analyzed in separate experiments the effect of cotransfecting THP-1 cells with the TNF-{alpha} promoter-luciferase reporter construct and the I{kappa}B superrepressor, an inhibitor of NF-{kappa}B activation (14). As expected, cotransfection with I{kappa}B superrepressor reduced TNF-{alpha} reporter gene activity by 77–87% in the 37°C cell cultures at time points from 2 to 24 h. The NF-{kappa}B inhibitor caused a comparable 75–88% reduction in TNF-{alpha} reporter plasmid activity in the 32°C cells at various time points, indicating that the augmented TNF-{alpha} expression in the hypothermic cells also was mediated, in part, by enhanced NF-{kappa}B-dependent TNF-{alpha} transcription.

Hypothermia prolonged NF-{kappa}B-responsive, but not AP-1-responsive, transactivation. Because NF-{kappa}B is known to play a major role in the activation of TNF transcription in LPS-stimulated monocytes, we analyzed the effect of hypothermia on the kinetics of NF-{kappa}B activation in LPS-stimulated THP-1 cells. After 30-min preincubation at 32 or 37°C, THP-1 cells were stimulated with 100 ng/ml LPS at the indicated temperature, and levels of active NF-{kappa}B in cell lysates were measured by EMSA (Fig. 6A, top). Baseline NF-{kappa}B activation after 30-min preincubation was comparably low in 32 and 37°C cell cultures. After the addition of LPS, there was a trend toward a delay in the initiation of NF-{kappa}B binding at 60 min that was not statistically significant. However, NF-{kappa}B activation was significantly prolonged at the lower temperature. Quantitation of the EMSA bands by laser densitometry (Fig. 6B) showed that levels of activated NF-{kappa}B in the 32°C cells were 60 and 67% greater than in the 37°C cells after 2- and 4-h LPS treatment, respectively. Supershift analysis showed the composition of the NF-{kappa}B complexes to be similar in the cells at both temperatures, suggesting that NF-{kappa}B dimer pairing is not significantly affected by moderate hypothermia. In contrast to the augmented NF-{kappa}B activation at 32°C, activation of the AP-1 family of proinflammatory transcription factors, as measured by gel shift, was comparable in 32 and 37°C cells (Fig. 6, A, bottom, and C).



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Fig. 6. Electrophoretic mobility shift assay (EMSA) of NF-{kappa}B and AP-1 activation in hypothermic vs. normothermic cell cultures. THP-1 cells were preincubated at 32 or 37°C for 30 min and treated with 100 ng/ml LPS at the respective temperatures for the indicated times. A: whole cell extracts were prepared, and EMSA was performed for activated NF-{kappa}B and AP-1. Representative blots of 3 separate experiments for each transcription factor are shown. Supershift was performed with antibodies to c-rel, p50, and p65 in cells treated with LPS for 1 h at 32 or 37°C (lanes 13–16). NF-{kappa}B (B) and AP-1 (C) band densities from 3 experiments were quantified by densitometry. Data represent means ± SE of 3 experiments. *P < 0.05, 32 vs. 37°C.

 
Reexpression of I{kappa}B{alpha} after LPS stimulation was delayed in hypothermic THP-1 cells. NF-{kappa}B activation is initiated by the targeted proteolysis of cytoplasmic I{kappa}B{alpha} and I{kappa}B{beta} and subsequent nuclear translocation NF-{kappa}B dimers. Once initiated, the expression of NF-{kappa}B-responsive genes is terminated, in part, by the reexpression and nuclear import of I{kappa}B{alpha} (1). We analyzed the effects of hypothermia on I{kappa}B{alpha} and I{kappa}B{beta} degradation and I{kappa}B{alpha} reappearance after LPS stimulation using Western blotting (Fig. 7) and Northern blotting (Fig. 8). Degradation of I{kappa}B{alpha} and I{kappa}B{beta} after LPS treatment was not significantly different at the two temperatures. However, whereas I{kappa}B{alpha} protein subsequently reappeared in both 32 and 37°C cell cultures, the reappearance was delayed by 2 h (Fig. 7; compare lanes 5–8) in the 32°C cells. By 4 h of LPS stimulation at 32°C, newly synthesized I{kappa}B{alpha} reached only one-half the level achieved in 37°C cells (Fig. 7; compare lanes 7 and 8). In contrast to the temperature-dependent reexpression of I{kappa}B{alpha}, I{kappa}B{beta} expression remained lower 4 h after LPS stimulation in cells at both 32 and 37°C. Stimulating THP-1 cells with LPS induced increases in I{kappa}B{alpha} mRNA levels in cells at both 32 and 37°C, but the increase occurred 30 min later (Fig. 8; compare lanes 3–6) and peak levels were delayed 1 h (Fig. 8; compare lanes 5–8) in the 32°C cells, indicating that I{kappa}B{alpha} gene expression is delayed in hypothermic cells.



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Fig. 7. Immunoblot analysis of I{kappa}B{alpha} and I{kappa}B{beta} expression in LPS-treated THP-1 cells incubated at 32 or 37°C. THP-1 cells were preincubated at 32 or 37°C for 30 min before addition of 100 ng/ml LPS. At indicated time points after addition of LPS, cell lysates were prepared and analyzed by Western blotting for I{kappa}B{alpha} (top) and I{kappa}B{beta} (bottom). Blots are representative of 3 separate experiments with similar results.

 


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Fig. 8. Northern blot analysis of I{kappa}B{alpha} expression in LPS-stimulated THP-1 cells incubated at 32 or 37°C. THP-1 cells were preincubated at 32 or 37°C for 30 min before addition of 100 ng/ml LPS. At the indicated time points after addition of LPS, cells were lysed, and total RNA was isolated and analyzed by Northern blotting for I{kappa}B{alpha}. Equivalent RNA loading was verified by UV visualization of ethidium bromide-stained 18S and 28S rRNA bands (bottom). The blot shown is representative of 3 experiments with similar results.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that in vitro exposure to moderate, clinically relevant hypothermia (32°C) delays and prolongs LPS-stimulated secretion of TNF-{alpha} and IL-1{beta} in primary cultures of human mononuclear phagocytes (28). In the current study, we used the THP-1 human promonocytic cell line model to extend these observations by showing that hypothermia delays onset but subsequently prolongs accumulation of TNF-{alpha} and IL-1{beta} mRNA at the level of gene transcription. We also provide novel data demonstrating delayed reexpression of the inhibitor I{kappa}B{alpha} at 32°C, accompanied by sustained activation of NF-{kappa}B, which may contribute to the prolonged cytokine production in hypothermic cells.

The hypothermia-induced changes TNF-{alpha} and IL-1{beta} secretion that we describe in THP-1 cells are similar to those that we (28) and others (45) have reported in primary cultured human monocytes and macrophages. Luhm et al. (45), using a human mixed peripheral blood mononuclear cell (PBMC) culture system, also found that hypothermia (30–34.5°C) increased the magnitude and duration of TNF-{alpha} and IL-1{beta} secretion. However, they found that hypothermia augmented cytokine secretion only in PBMC stimulated with LPS, purified lipid A, or gram-negative bacteria, and not in PBMC stimulated with structurally dissimilar agents, including TSST-1. They concluded that hypothermia augments LPS-induced cytokine generation by increasing LPS bioactivity rather than by modifying the cellular response to stimulation. In contrast, we found that hypothermia exerted similar effects on cytokine generation induced by LPS and two structurally distinct stimuli, opsonized zymosan and TSST-1, suggesting that hypothermia augments cytokine generation by modifying the monocyte cellular response to diverse stimuli. We excluded the potential contribution of contaminating LPS by confirming that the TSST-1 preparation did not contain sufficient endotoxin activity to activate THP-1 cells in the presence of polymyxin B. In contrast to the pure monocyte cell culture system used in the present study, Luhm et al. (45) analyzed a mixed PBMC cell culture system containing both monocytes and lymphocytes. Because monocytes express cell surface CD14, they are highly responsive to stimulation by LPS (22), but we found that TSST-1 was only a weak activator of cytokine release in THP-1 monocytes. In contrast, T lymphocytes are highly responsive to TSST-1, which acts by binding to a nonpolymorphic domain on the T-cell receptor (42). The failure of Luhm et al. (45) to show comparable temperature dependence of LPS- and TSST-1-induced cytokine generation in the PBMC culture system may thus reflect cell-specific differences in the hypothermic response.

Moderate hypothermia has been shown to increase the expression of a small number of genes in mammalian cells, including the gene for cold-inducible RNA binding protein (50), immediate early genes in the fos and jun families (40), and the antiapoptotic protein Bcl-x (49), and our studies add TNF-{alpha} and IL-1{beta} to this list of cold-inducible genes. We provide two lines of evidence showing that alterations in TNF-{alpha} gene transcription, as opposed to transcript stability or translation, are responsible for prolonged cytokine generation in hypothermic cells. First, TNF-{alpha} mRNA was not sufficiently stabilized at 32°C to account for the marked increase in TNF-{alpha} mRNA accumulation. Because transcript levels are determined by transcription and mRNA degradation kinetics (52), these results imply that hypothermia prolongs TNF-{alpha} transcription, a conclusion further supported by the effects of hypothermia on TNF-{alpha} reporter plasmid activity. We used a dual luciferase reporter gene system in which experimental and control plasmids generate firefly luciferase and Renilla luciferase, respectively. In preliminary experiments, the activities of firefly luciferase and Renilla luciferase plasmids, driven by the same thymidine kinase promoter, were similar in 32 and 37°C cells, indicating that posttranscriptional processing of the two reporter genes was temperature independent within the range studied. The 1.1-kb TNF-{alpha} 5' flanking sequence contains regulatory regions conferring responsiveness to a variety of stimuli, including LPS (59), phorbol esters (24), and UV light (5). NF-{kappa}B binding sites in the TNF-{alpha} promoter have been shown to be required for maximal LPS-induced TNF-{alpha} transcription in monocytes. In the present study, we confirmed the NF-{kappa}B dependence of TNF-{alpha} transcription in LPS-stimulated THP-1 cells by showing that cotransfection with an inhibitor of NF-{kappa}B activation, the I{kappa}B{alpha} superrepressor (14), inhibits NF-{kappa}B reporter plasmid activity by as much as 88% in 37°C cell cultures. Importantly, cotransfection with I{kappa}B{alpha} superrepressor comparably inhibited TNF-{alpha} reporter gene activity in 32 and 37°C cell cultures, indicating that excess TNF-{alpha} transcriptional activity at 32°C also was NF-{kappa}B dependent.

Our gel shift analysis showed that LPS-induced NF-{kappa}B activation was prolonged in the 32°C cells compared with the 37°C cells. In contrast, LPS induced comparable activation of AP-1 at the two temperatures. This suggests that hypothermia modifies signaling pathways distal to the recruitment of TNF receptor-associated factor 6 to the TLR4/MyD88 receptor complex, where the signaling pathways leading to activation of NF-{kappa}B and AP-1 bifurcate (69). Other groups (35, 41, 61) have studied the effects of moderate in vitro or in vivo hypothermia on NF-{kappa}B activation, with both diminished and augmented activation reported. Sutcliffe et al. (61) reported that exposing cultured human cerebral endothelial cells to moderate hypothermia reduced NF-{kappa}B-dependent reporter activity measured 4 h after stimulation with IL-1{beta}, but other time points were not studied. In a rat model of bacterial meningitis, Irazuzta et al. (35) found that 6-h exposure to hypothermia (32–34°C) was associated with a 32% reduction in NF-{kappa}B activation in cerebral nuclei compared with meningitic animals that remained euthermic. Although we did not find a consistent delay in the onset of NF-{kappa}B activation at 32°C, we found significantly enhanced activation at 2 and 4 h after LPS stimulation. Our findings are similar to those of Kimura et al. (41), who showed that in LPS-stimulated PBMC, moderate hypothermia (33°C) delayed onset of NF-{kappa}B activation by 30 min and TNF-{alpha} generation by 1 h but increased levels of NF-{kappa}B activation after 2 h and TNF-{alpha} expression after 24 h. It is likely that hypothermia differentially affects NF-{kappa}B activation and cytokine generation, depending on the degree and duration of hypothermia, the stimulus used, and the cells, organs, and species under investigation.

The sequence of events leading to termination of NF-{kappa}B activation is critically important to host defense, because overexpression of inflammatory cytokines plays a role in the pathology of sepsis and other inflammatory diseases. NF-{kappa}B activation is temporally limited, in part, through reexpression of I{kappa}B{alpha} (1), which is itself an NF-{kappa}B-responsive gene. We provide novel evidence that after LPS-induced degradation of the I{kappa}B complex, reexpression of I{kappa}B{alpha} is delayed by several hours under hypothermic conditions at both the mRNA and protein levels. Such a delay in activation of this counterregulatory pathway may explain, in part, the prolonged expression of NF-{kappa}B-responsive genes, including TNF-{alpha} and IL-1{beta}, that occurs in hypothermic cells.

Our findings that moderate hypothermia prolongs NF-{kappa}B activation and alters cytokine gene expression may provide a partial explanation for the high morbidity and mortality associated with hypothermia in some clinical settings. A recent, large clinical trial (3) found elevated serum TNF-{alpha} and IL-6 levels and twofold higher mortality in adults with sepsis who were hypothermic compared with adults with sepsis who had fever. Rather than being simply a marker for more severe sepsis, low body temperature may result in prolonged production of inflammatory cytokines, leading to endothelial cell damage, capillary leak, shock, and multiple organ dysfunction. Even in clinical situations not associated with LPS exposure, such as accidental hypothermia, anesthesia-related intraoperative hypothermia, and hypothermic circulatory arrest during cardiac surgery, core temperatures in the 28–35°C range have been associated with altered cytokine production (6), impaired host defense (43), and increased mortality (3, 16, 39, 46). Further understanding of the effects of hypothermia on the regulation of NF-{kappa}B activation and the counterregulatory mechanisms that limit this process will improve our ability to predict the effects of hypothermia on inflammation in the infected or injured host.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by American Heart Association Grant 0365547U (to K. D. Fairchild), National Institute of Allergy and Infectious Diseases Grant AI-42117, a Veterans Affairs Merit Review Award, and a Passano Foundation Physician-Scientist Award (to J. D. Hasday).


    ACKNOWLEDGMENTS
 
We thank Dr. Dhan Kalvakolanu for insight and encouragement.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. D. Fairchild, Div. of Neonatology, Rm. N5W68, Univ. of Maryland Hospital, 22 S. Greene St., Baltimore, MD 21201 (E-mail: kfairchild{at}peds.umaryland.edu).

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. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Arenzana-Seisdedos F, Thompson J, Rodriguez MS, Bachelerie F, Thomas D, and Hay RT. Inducible nuclear expression of newly synthesized I{kappa}B{alpha} negatively regulates DNA-binding and transcriptional activities of NF-{kappa}B. Mol Cell Biol 15: 2689–2696, 1995.[Abstract]

2. Armstrong C. Some recent research in the field of neurotropic viruses with especial reference to lymphocytic choriomeningitis and herpes simplex. Mil Surg 91: 129–145, 1942.

3. Arons MM, Wheeler AP, Bernard GR, Christman BW, Russell JA, Schein R, Summer WR, Steinberg KP, Fulkerson W, Wright P, Dupont WD, and Swindell BB. Effects of ibuprofen on the physiology and survival of hypothermic sepsis. Ibuprofen in Sepsis Study Group. Crit Care Med 27: 699–707, 1999.[ISI][Medline]

4. Baeuerle P and Baltimore D. NF-{kappa}B: ten years after. Cell 87: 13–20, 1996.[ISI][Medline]

5. Bazzoni F, Kruys V, Shakhov A, Jongeneel CV, and Beutler B. Analysis of tumor necrosis factor promoter responses to ultraviolet light. J Clin Invest 93: 56–62, 1994.[ISI][Medline]

6. Beilin B, Shavit Y, Razumovsky J, Wolloch Y, Zeidel A, and Bessler H. Effects of mild perioperative hypothermia on cellular immune responses. Anesthesiology 89: 1133–1140, 1998.[ISI][Medline]

7. Belasco JG and Brawerman G. Experimental approaches to the study of mRNA decay. In: Control of Messenger RNA Stability, edited by Belasco JG and Brawerman G. San Diego, CA: Academic, 1993, p. 475–493.

8. Bernard SA, Gray TW, Buist MD, Jones BM, Silvester W, Gutteridge G, and Smith K. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 346: 557–563, 2002.[Abstract/Free Full Text]

9. Bernheim HA and Kluger MJ. Fever: effect of drug-induced antipyresis on survival. Science 193: 237–239, 1976.[ISI][Medline]

10. Beutler B, Krochin N, Milsark IW, Luedke C, and Cerami A. Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science 232: 977–979, 1986.[ISI][Medline]

11. Beutler B, Milsark I, and Cerami A. Passive immunization against cachectin/tumor necrosis factor protects mice from the lethal effect of endotoxin. Science 229: 869–871, 1985.[ISI][Medline]

12. Bierens JJ, Uitslager R, Swenne-van Ingen MM, van Stiphout WA, and Knape JT. Accidental hypothermia: incidence, risk factors and clinical course of patients admitted to hospital. Eur J Emerg Med 2: 38–46, 1995.[Medline]

13. Brown GD, Herre J, Williams DL, Willment JA, Marshall AS, and Gordon S. Dectin-1 mediates the biological effects of {beta}-glucans. J Exp Med 197: 1119–1124, 2003.[Abstract/Free Full Text]

14. Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, and Maniatis T. Signal-induced site-specific phosphorylation targets I{kappa}B{alpha} to the ubiquitin-proteasome pathway. Genes Dev 9: 1586–1597, 1995.[Abstract]

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

16. Clemmer TP, Fisher CJ Jr, Bone RC, Slotman GJ, Metz CA, and Thomas FO. Hypothermia in the sepsis syndrome and clinical outcome. The Methylprednisolone Severe Sepsis Study Group. Crit Care Med 20: 1395–1401, 1992.[ISI][Medline]

17. Covert JB and Reynolds WW. Survival value of fever in fish. Nature 267: 43–45, 1977.[ISI][Medline]

18. Cross A, Asher L, Seguin M, Yuan L, Kelly N, Hammack C, Sadoff J, and Gemski P Jr. The importance of a lipopolysaccharide-initiated, cytokine-mediated host defense mechanism in mice against extraintestinally invasive Escherichia coli. J Clin Invest 96: 676–686, 1995.[ISI][Medline]

19. Cross AS, Sadoff JC, Kelly N, Bernton E, and Gemski P. Pretreatment with recombinant murine tumor necrosis factor {alpha}/cachectin and murine interleukin 1{alpha} protects mice from lethal bacterial infection. J Exp Med 169: 2021–2027, 1989.[Abstract]

20. Dinarello CA. Cytokines as endogenous pyrogens. In: Fever: Basic Mechanisms and Management (2nd ed.), edited by Mackowiak PA. New York: Raven, 1996, p. 87–116.

21. Doherty GM, Lange JR, Langstein HN, Alexander HR, Buresh CM, and Norton JA. Evidence for IFN-{gamma} as a mediator of the lethality of endotoxin and tumor necrosis factor-{alpha}. J Immunol 149: 1666–1670, 1992.[Abstract/Free Full Text]

22. Drouet C, Shakhov AN, and Jongeneel CV. Enhancers and transcription factors controlling the inducibility of the tumor necrosis factor-{alpha} promoter in primary macrophages. J Immunol 147: 1694–1700, 1991.[Abstract/Free Full Text]

23. Dubin WL, Martin TR, Swoveland P, Leturcq DJ, Moriarty AM, Tobias PS, Bleecker ER, Goldblum SE, and Hasday JD. Asthma and endotoxin: lipopolysaccharide-binding protein and soluble CD14 in bronchoalveolar compartment. Am J Physiol Lung Mol Cell Physiol 270: L736–L744, 1996.[Abstract/Free Full Text]

24. Economou JS, Rhoades K, Essner R, McBride WH, Gasson JC, and Morton DL. Genetic analysis of the human tumor necrosis factor {alpha}/cachectin promoter region in a macrophage cell line. J Exp Med 170: 321–326, 1989.[Abstract]

25. Elstad MR, Parker CJ, Cowley FS, Wilcox LA, McIntyre TM, Prescott SM, and Zimmerman GA. CD11b/CD18 integrin and a {beta}-glucan receptor act in concert to induce the synthesis of platelet-activating factor by monocytes. J Immunol 152: 220–230, 1994.[Abstract/Free Full Text]

26. Ensor JE, Crawford EK, and Hasday JD. Warming macrophages to febrile range destabilizes tumor necrosis factor-{alpha} mRNA without inducing heat shock. Am J Physiol Cell Physiol 269: C1140–C1146, 1995.[Abstract/Free Full Text]

27. Ensor JE, Wiener SM, McCrea KA, Viscardi RM, Crawford EK, and Hasday JD. Differential effects of hyperthermia on macrophage interleukin-6 and tumor necrosis factor-{alpha} expression. Am J Physiol Cell Physiol 266: C967–C974, 1994.[Abstract/Free Full Text]

28. Fairchild KD, Viscardi RM, Hester L, Singh IS, and Hasday JD. Effects of hypothermia and hyperthermia on cytokine production by cultured human mononuclear phagocytes from adults and newborns. J Interferon Cytokine Res 20: 1049–1055, 2000.[CrossRef][ISI][Medline]

29. Godambe SA, Chaplin DD, and Bellone CJ. Regulation of IL-1 gene expression: differential responsiveness of murine macrophage lines. Cytokine 5: 327–335, 1993.[ISI][Medline]

30. Greenberg ME and Belasco JG. Control of the decay of labile protooncogene and cytokine mRNAs. In: Control of Messenger RNA Stability, edited by Belasco JG and Brawerman G. San Diego, CA: Academic, 1993, p. 199–218.

31. Han J, Brown T, and Beutler B. Endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level. J Exp Med 171: 465–475, 1990.[Abstract]

32. Hasday JD, Fairchild KD, and Shanholtz C. The role of fever in the infected host. Microbes Infect 2: 1891–1904, 2000.[CrossRef][ISI][Medline]

33. Hasday JD and Singh IS. Fever and the heat shock response: distinct, partially overlapping processes. Cell Stress Chaperones 5: 471–480, 2000.[ISI][Medline]

34. Hentze MW. Determinants and regulation of cytoplasmic mRNA stability in eukaryotic cells. Biochim Biophys Acta 1090: 281–292, 1991.[ISI][Medline]

35. Irazuzta JE, Pretzlaff RK, Zingarelli B, Xue V, and Zemlan F. Modulation of nuclear factor-{kappa}{beta} activation and decreased markers of neurological injury associated with hypothermic therapy in experimental bacterial meningitis. Crit Care Med 30: 2553–2559, 2002.[CrossRef][ISI][Medline]

36. Jiang Q, Cross AS, Singh IS, Chem TT, Viscardi RM, and Hasday JD. Febrile core temperature is essential for optimal host defense in bacterial peritonitis. Infect Immun 68: 1265–1270, 2000.[Abstract/Free Full Text]

37. Jiang Q, Detolla L, Singh IS, Gatdula L, Fitzgerald B, van Rooijen N, Cross AS, and Hasday JD. Exposure to febrile temperature upregulates expression of pyrogenic cytokines in endotoxin-challenged mice. Am J Physiol Regul Integr Comp Physiol 276: R1653–R1660, 1999.[Abstract/Free Full Text]

38. Jongeneel CV, Shakhov AN, Nedospasov SA, and Cerottini JC. Molecular control of tissue-specific expression at the mouse TNF locus. Eur J Immunol 19: 549–552, 1989.[ISI][Medline]

39. Jurkovich GJ, Greiser WB, Luterman A, and Curreri PW. Hypothermia in trauma victims: an ominous predictor of survival. J Trauma 27: 1019–1024, 1987.[ISI][Medline]

40. Kamme F, Campbell K, and Wieloch T. Biphasic expression of the fos and jun families of transcription factors following transient forebrain ischaemia in the rat. Effect of hypothermia. Eur J Neurosci 7: 2007–2016, 1995.[ISI][Medline]

41. Kimura A, Sakurada S, Ohkuni H, Todome Y, and Kurata K. Moderate hypothermia delays proinflammatory cytokine production of human peripheral blood mononuclear cells. Crit Care Med 30: 1499–1502, 2002.[ISI][Medline]

42. Kum WW, Cameron SB, Hung RW, Kalyan S, and Chow AW. Temporal sequence and kinetics of proinflammatory and anti-inflammatory cytokine secretion induced by toxic shock syndrome toxin 1 in human peripheral blood mononuclear cells. Infect Immun 69: 7544–7549, 2001.[Abstract/Free Full Text]

43. Kurz A, Sessler DI, and Lenhardt R. Perioperative normothermia to reduce the incidence of surgical-wound infections and shorten hospitalization. N Engl J Med 334: 1209–1215, 1996.[Abstract/Free Full Text]

44. Lieberman AP, Pitha PM, and Shin ML. Protein kinase regulates tumor necrosis factor mRNA stability in virus-stimulated astrocytes. J Exp Med 172: 989–992, 1990.[Abstract]

45. Luhm J, Schromm AB, Seydel U, Brandenburg K, Wellinghausen N, Riedel E, Schumann RR, and Rink L. Hypothermia enhances the biological activity of lipopolysaccharide by altering its fluidity state. Eur J Biochem 256: 325–333, 1998.[Abstract]

46. Luna GK, Maier RV, Pavlin EG, Anardi D, Copass MK, and Oreskovich MR. Incidence and effect of hypothermia in seriously injured patients. J Trauma 27: 1014–1018, 1987.[ISI][Medline]

47. Mathur NB, Singh A, Sharma VK, and Satyanarayana L. Evaluation of risk factors for fatal neonatal sepsis. Indian Pediatr 33: 817–822, 1996.[Medline]

48. Mosser DD, Theodorakis NG, and Morimoto RI. Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells. Mol Cell Biol 8: 4736–4744, 1988.[ISI][Medline]

49. Ning XH, Chen SH, Xu CS, Li L, Yao LY, Qian K, Krueger JJ, Hyyti OM, and Portman MA. Hypothermic protection of the ischemic heart via alterations in apoptotic pathways as assessed by gene array analysis. J Appl Physiol 92: 2200–2207, 2002.[Abstract/Free Full Text]

50. Nishiyama H, Higashitsuji H, Yokoi H, Itoh K, Danno S, Matsuda T, and Fujita J. Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene. Gene 204: 115–120, 1997.[CrossRef][ISI][Medline]

51. Ozinsky A, Underhill DM, Fontenot JD, Hajjar AM, Smith KD, Wilson CB, Schroeder L, and Aderem A. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc Natl Acad Sci USA 97: 13766–13771, 2000.[Abstract/Free Full Text]

52. Raabe T, Bukrinsky M, and Currie RA. Relative contribution of transcription and translation to the induction of tumor necrosis factor-{alpha} by lipopolysaccharide. J Biol Chem 273: 974–980, 1998.[Abstract/Free Full Text]

53. Roberts JR, Rowe PA, and Demaine AG. Activation of NF-{kappa}B and MAP kinase cascades by hypothermic stress in endothelial cells. Cryobiology 44: 161–169, 2002.[CrossRef][ISI][Medline]

54. Saccani S, Pantano S, and Natoli G. Two waves of nuclear factor {kappa}B recruitment to target promoters. J Exp Med 193: 1351–1359, 2001.[Abstract/Free Full Text]

55. Sariban E, Imamura K, Luebbers R, and Kufe D. Transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes. J Clin Invest 81: 1506–1510, 1988.[ISI][Medline]

56. Schmidt JR and Rasmussen AF Jr. The influence of environmental temperature on the course of experimental herpes simplex infection. J Infect Dis 107: 356–360, 1960.[ISI]

57. Schreiber E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding protein with mini extracts prepared from a small number of cells. Nucleic Acids Res 17: 6419–0000, 1989.[ISI][Medline]

58. Schwende H, Fitzke E, Ambs P, and Dieter P. Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. J Leukoc Biol 59: 555–561, 1996.[Abstract]

59. Shakhov AN, Collart MA, Vassalli P, Nedospasov SA, and Jongeneel CV. {kappa}B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor {alpha} gene in primary macrophages. J Exp Med 171: 35–47, 1990.[Abstract]

60. Singh IS, Calderwood S, Kalvakolanu I, Viscardi R, and Hasday JD. Inhibition of tumor necrosis factor-{alpha} transcription in macrophages exposed to febrile range temperature: a possible role for heat shock factor-1. J Biol Chem 275: 9841–9848, 2000.[Abstract/Free Full Text]

61. Sutcliffe IT, Smith HA, Stanimirovic D, and Hutchison JS. Effects of moderate hypothermia on IL-1 {beta}-induced leukocyte rolling and adhesion in pial microcirculation of mice and on proinflammatory gene expression in human cerebral endothelial cells. J Cereb Blood Flow Metab 21: 1310–1319, 2001.[ISI][Medline]

62. Taffet SM, Singhel KJ, Overholtzer JF, and Shurtleff SA. Regulation of tumor necrosis factor expression in a macrophage-like cell line by lipopolysaccharide and cyclic AMP. Cell Immunol 120: 291–300, 1989.[ISI][Medline]

63. Tam W, Lee L, Davis L, and Sen R. Cytoplasmic sequestration of rel proteins by I{kappa}B{alpha} requires CRM1-dependent nuclear export. Mol Cell Biol 20: 2269–2284, 2000.[Abstract/Free Full Text]

64. Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S, Mizuno T, and Tohyama M. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF{kappa}B activation in primary hippocampal neurons. J Biol Chem 274: 8531–8538, 1999.[Abstract/Free Full Text]

65. Tracey KJ, Wei H, Manogue KR, Fong Y, Hesse DG, Nguyen HT, Kuo GC, Beutler B, Cotran RS, Cerami A, and Lowry SF. Cachectin/tumor necrosis factor induces cachexia, anemia, and inflammation. J Exp Med 167: 1211–1227, 1988.[Abstract]

66. Tsai EY, Falvo JV, Tsytsykova AV, Barczak AK, Reimold AM, Glimcher LH, Fenton MJ, Gordon DC, Dunn IF, and Goldfeld AE. A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor {alpha} promoter in vivo. Mol Cell Biol 20: 6084–6094, 2000.[Abstract/Free Full Text]

67. Waage A and Espevik T. Interleukin 1 potentiates the lethal effect of tumor necrosis factor {alpha}/cachectin in mice. J Exp Med 167: 1987–1992, 1988.[Abstract]

68. Webb P. Temperatures of skin, subcutaneous tissue, muscle and core in resting men in cold, comfortable and hot conditions. Eur J Appl Physiol 64: 471–476, 1992.

69. Zhang FX, Kirschning CJ, Mancinelli R, Xu XP, Jin Y, Faure E, Mantovani A, Rothe M, Muzio M, and Arditi M. Bacterial lipopolysaccharide activates nuclear factor-{kappa}B through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J Biol Chem 274: 7611–7614, 1999.[Abstract/Free Full Text]