1Department of Pediatrics, University of Virginia Children's Hospital, Charlottesville, Virginia; Departments of 2Pediatrics and 3Medicine, University of Maryland School of Medicine; 4University of Maryland at Baltimore Cytokine Core Laboratory; and 5Medicine and Research Services of the Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
Submitted 1 April 2005 ; accepted in final form 10 June 2005
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ABSTRACT |
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tumor necrosis factor; monocytes
Spontaneous HT has been associated with poor outcome in a number of clinical settings, including sepsis, in which patients with hypothermia have an approximately twofold higher mortality than those presenting with fever (3, 25) and in severe trauma, in which mortality is greatly increased in patients with HT upon arrival at the hospital (23). Studies from several laboratories have shown that HT may impair host defense mechanisms (8, 26). Our own studies have shown that clinically relevant HT delays the onset but increases the duration and magnitude of inflammatory cytokine generation, in part through effects on the transcription factor NF-B (13). While an initial lag in TNF-
production may delay activation of innate host defenses, the sustained production of TNF-
with continued exposure to HT might lead to increased tissue injury and contribute to a worse outcome in certain clinical situations. On the other hand, enhanced expression of other genes regulated by NF-
B, including multiple antiapoptotic proteins, may confer cytoprotection and improve clinical outcome in other conditions.
Despite widespread interest in clinical applications of HT, relatively little is known about its cellular and molecular effects. In the present study, we have analyzed the effect of moderate and marked HT on the biochemical events that activate and subsequently terminate NF-B-dependent transcription in LPS-challenged human THP-1 promonocytic cells. Specifically, we have analyzed the effects of HT on the classic pathway of NF-
B activation in which IKK-
phosphorylates the inhibitory I
B-
, targeting it for polyubiquitination and proteasomal degradation. We also analyzed the effects of HT on the reexpression of I
B-
, an important autoregulatory negative feedback mechanism that terminates NF-
B-dependent transcription, thereby reducing the duration of NF-
B-dependent gene expression (2).
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MATERIALS AND METHODS |
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ELISA.
Human TNF- levels were measured at the University of Maryland at Baltimore Cytokine Core Laboratory by performing two-antibody ELISA with biotin-streptavidin-peroxidase detection using commercially available antibodies and recombinant standard (Biosource, Camarillo, CA) as previously described (14). TNF-
concentrations were calculated from a recombinant human TNF-
standard curve using a computer program (Softmax; Molecular Devices). The assay had a lower limit of detection of 15 pg/ml. To analyze effects of HT on the clearance of TNF-
from culture medium by THP-1 cells, we sequentially analyzed levels of exogenous recombinant murine TNF-
(Pierce/Endogen, Boston, MA) in the culture medium by performing ELISA using an antibody pair that does not cross-react with human TNF-
(BioSource).
Western blot analysis.
Cells were cultured at a density of 1 x 106/ml and exposed to LPS and hypothermic or normothermic culture temperatures as described for the analysis of TNF- secretion. For Western blot analysis of NF-
B signaling pathway components, cells were lysed in RIPA buffer [10 mM Tris, pH 8, 100 mM NaCl, 1% Nonidet P-40 (NP-40), and 0.1% SDS]. For analysis of nuclear RelA/p65 expression, nuclear extracts were prepared by suspending cells in buffer A (in mM: 10 HEPES, 10 KCl, 0.1 EDTA, 0.1 EGTA, and 0.1 DTT) on ice for 15 min, followed by addition of 10% NP-40. After centrifugation, the nuclear pellets were resuspended in buffer C (in mM: 20 HEPES, pH 7.9, 400 NaCl, 1 EDTA, 1 EGTA, and 1 DTT) for 30 min with shaking at 4°C. Protease inhibitors (Complete Mini kit; Roche Molecular Biochemicals, Indianapolis, IN) and phosphatase inhibitors (Phosphatase Cocktail I and II; Sigma) were added to RIPA buffer and to buffers A and C. The protein content of all samples was quantitated using a commercial reagent based on the Bradford reaction with a bovine serum albumin standard curve. For Western blot analysis, sample aliquots containing 10 µg of protein were separated on 10% SDS-polyacrylamide gels and then electrostatically transferred to PVDF membranes (Immobilon; Millipore, Bedford, MA). After being blocked with 5% nonfat milk, membranes were probed with one of the following antibodies at 1:1,000 dilution unless otherwise indicated: I
B-
, IKK-
, phospho-IKK-
/IKK-
S180/S181, RelA/p65 (all obtained from Cell Signaling Technology, Beverly, MA), heat shock proteins (HSPs) 70 and/or 72 at 1:10,000 dilution (Stressgen),
-tubulin (Chemicon International, Temecula, CA), and actin (Sigma). Bands were detected using a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody (Sigma) and an enhanced chemiluminescence detection system (ECL Plus; Amersham Biosciences). Membranes were exposed to X-ray film, and the band intensity was quantified by performing densitometric analysis (Molecular Dynamics, Sunnyvale, CA) with results in each experiment normalized to baseline time 0 at 37°C (no LPS) band density. Results are expressed as the relative increase in band density in hypothermic vs. normothermic cells for each experimental condition.
EMSA.
For EMSA analysis, cells were cultured as described for Western blot analysis, extracts were prepared according to the method described by Schreiber et al. (30), and EMSA was performed as previously described (32). A double-stranded oligonucleotide containing the heat shock response element (HRE) corresponding to 107/83 of the human HSP70 promoter 5'-GATCTCGGCTGGAATATTCCCGACCTGGCAGCCGA-3', and the complementary strands were synthesized, annealed, and [-32P]ATP labeled using T4 polynucleotide kinase (Promega; Madison, WI) according to the manufacturer's protocol. Cell extracts and probe 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 of poly(dI-dC). Samples were separated on 4% polyacrylamide gels, and after being dried, gels were exposed to X-ray film.
Statistics. All data represent at least three independent experiments performed on different days. Data are expressed as means ± SE unless otherwise indicated. Differences between temperatures at multiple time points were examined using ANOVA, or, where appropriate, ANOVA on ranks with multiple-comparison testing using the Student-Newman-Keuls method with SigmaStat for Windows software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered statistically significant.
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RESULTS |
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We previously showed that vitamin D3-differentiated THP-1 cells produced three- to fivefold more TNF- and IL-1
than undifferentiated THP-1 cells upon stimulation with LPS but that HT exerts comparable effects on cytokine production in differentiated and undifferentiated cells. In all experiments in the present study, we used vitamin D3-differentiated THP-1 cells. Treatment with 100 ng/ml LPS at 37°C stimulated a rapid release of TNF-
, with levels in culture supernatants peaking by 2 h and declining to 38% of peak levels by 24 h (Fig. 1). Under hypothermic conditions, the rise in TNF-
levels after addition of LPS was delayed, with peak levels not attained until 6 h (data not shown) and higher levels persisting 24 h after stimulation with LPS. Under conditions of marked (28°C) and moderate (32°C) HT, TNF-
levels 2 h after addition of LPS were 27% and 77% of levels in the 37°C cultures (P < 0.05, 28°C vs. 37°C). However, peak TNF-
levels were 20% and 22% higher at 28°C and 32°C vs. 37°C, and by 24 h, TNF-
levels were 3.1- and 2.5-fold higher in the 28°C and 32°C culture supernatants, respectively, compared with the same time points in the 37°C cultures. Whereas TNF-
levels in the 37°C cultures rapidly declined between 6 and 24 h, there was a smaller decline during the same time span in the 32°C cultures. In the 28°C cultures, TNF-
levels were comparable in the 6- and 24-h culture supernatants.
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To determine whether HT might affect the stability of TNF- protein or reduce its uptake by cultured THP-1 cells, we added 100 pg/ml recombinant murine TNF-
to LPS-stimulated THP-1 cells subsequently cultured at 28°C, 32°C, or 37°C for 24 h and then analyzed the residual TNF-
using a species-specific murine TNF-
ELISA. After 24 h, culture supernatants from cells exposed to each of the three temperatures contained similar concentrations of murine TNF-
(data not shown), suggesting that the observed increase in TNF-
protein levels in the 28°C and 32°C culture supernatants was not caused by stabilization of TNF-
protein or alterations in cellular uptake of TNF-
at hypothermic temperatures. We also demonstrated that culturing THP-1 cells for 24 h at 28°C, 32°C, and 37°C did not significantly alter cell viability as measured using the following assays (n = 4 for each assay): Trypan blue dye exclusion (mean viability, 88%, 98%, and 90% at 28°C, 32°C, and 37°C, respectively), neutral red uptake [mean optical density (OD), 0.111, 0.105, and 0.110 at 28°C, 32°C, and 37°C, respectively], and reduction of 3-(4,5-dimethylthiazol-2-yl-5)-(3-carboxymethoxyphenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium, a marker of mitochondrial function (mean OD, 0.868, 1.152, and 1.181 at 28°C, 32°C, and 37°C, respectively; P = 0.117 at 28°C vs. 37°C).
Short-duration HT followed by rewarming augments TNF- production.
While we showed that sustained exposure to HT increases the duration and magnitude of TNF-
expression, such exposure in patients with accidental HT or sepsis is usually much briefer, because rewarming therapies are initiated. We therefore analyzed the effects of short-duration exposure to HT followed by rewarming on TNF-
production in THP-1 cells. Cells were stimulated with 1 or 10 ng/ml LPS or medium alone (control) for 2 h at 28°C, 32°C, or 37°C (Fig. 2). The culture medium was then changed, all cultures were switched to 37°C for 22 h with no LPS in the medium, and TNF-
levels in culture supernatants were measured using ELISA. Transient exposure to HT caused a qualitatively similar but more pronounced increase in TNF-
levels in the 24-h post-LPS culture supernatants than did sustained exposure to HT. Exposing cells to 10 ng/ml LPS at 28°C or 32°C for only 2 h followed by 22-h incubation at 37°C without LPS was sufficient to increase the levels of secreted TNF-
by 18.4- and 3.7-fold, respectively, compared with cells exposed to LPS for 2 h at 37°C.
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DISCUSSION |
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The exposure temperatures used in this study are commonly encountered in the clinical setting. Intentional reduction of core body temperature to 2028°C has been a traditional approach to minimizing myocardial ischemia-reperfusion injury during cardiac surgery (22), while more moderate HT with core temperatures between 32° and 34°C has been shown in both animal and human studies to confer neuroprotection in the setting of ischemia-reperfusion injury. Therapeutic HT to 3234°C for 1224 h after cardiac arrest has been shown to improve neurological outcomes in randomized, controlled trials (5, 20), and recent randomized trials in asphyxiated newborns have suggested that the induction of HT for 4872 h reduces neurological morbidity (12, 15). Enthusiasm for the benefits of HT in some clinical settings must be tempered, however, by concerns regarding possible adverse effects. For example, in a recent large study of HT during surgery for intracranial aneurysms, HT not only did not improve outcomes but also significantly increased the risk of postoperative bacteremia (33). The present study focused on the effects of HT on cytokine expression by the mononuclear phagocyte, a central regulator of the innate immune response. While the net effects of HT in intact animals is much more complex, understanding the effects of HT at the cellular level will allow a better understanding and more accurate predictions regarding how HT will modify patient outcomes in different clinical situations.
Although we have shown that TNF- production is initially delayed under hypothermic conditions, longer duration of HT significantly increases TNF-
levels in supernatants of THP-1 cells stimulated with low doses of LPS. We excluded stabilization of TNF-
protein in culture medium or the reduction of TNF-
clearance by THP-1 cells as contributing significantly to the enhanced accumulation in the hypothermic culture supernatants by analyzing the clearance rate of exogenous murine TNF-
from THP-1 cell-conditioned medium using a species-specific ELISA as previously described (32). Furthermore, we previously demonstrated directly that HT augments the activity of a TNF-
promoter-driven reporter construct and accumulation of TNF-
mRNA, indicating that HT augments TNF-
expression at least in part through an increase in TNF-
gene transcription (13). Because of the recognized central role of NF-
B in regulating transcriptional activation of proinflammatory cytokine genes, including TNF-
, we analyzed the effect of HT on activation of this transcription factor.
We have shown two important effects of HT on the kinetics of NF-B activation and TNF-
expression. Our previous studies showed a trend toward delayed activation of NF-
B by
1 h in LPS-stimulated cells exposed to 32°C vs. 37°C, mirrored by a brief delay in activation of NF-
B-dependent genes. This was followed at 1.54 h by increased activation of NF-
B in the hypothermic cells as measured using gel shift assay (14). In the present study, we have begun to uncover the mechanism of the HT effect by showing that phosphorylation of the NF-
B-activating I
B kinase is augmented and nuclear retention of p65 is prolonged during exposure to 28°C or 32°C culture conditions. It thus appears that HT alters both cytosolic and nuclear events, leading to prolonged duration of NF-
B activation and sustained expression of proinflammatory cytokines. Such a prolongation of cytokine gene expression increases the likelihood of simultaneous expression of cytokines that have additive cytotoxicity, such as TNF-
and IL-1
(34) or TNF-
and IFN-
(11). Furthermore, the capacity of HT to both delay initiation of NF-
B activation and forestall its subsequent inactivation explains the apparent discrepancy between the results of our studies and those reported by others that have shown a reduction in NF-
B activation and proinflammatory cytokine generation during short-term exposure to HT. For example, Han et al. (15) showed in a rat stroke model that maintaining core temperature at 33°C vs. 37°C for 2 h decreased NF-
B nuclear translocation and expression of TNF-
and inducible NO synthase in brain tissue. In a rat model of endotoxemia, Skumpia et al. (31) showed that 150 min of severe HT (1824°C) decreased NF-
B activation and IL-1
expression in the heart. In clinical settings, therapeutic HT in the 3234°C range is continued for up to several days; thus it is important to extend the duration of HT in both in vitro and in vivo studies to better understand the mechanisms of both beneficial and detrimental effects of this therapy.
In pursuing the mechanism of prolonged NF-B activation during HT, we have shown that I
B-
reexpression in LPS-treated THP-1 cells is delayed in cells cultured at 28°C or 32°C compared with those cultured at 37°C. Reexpression of I
B-
has been shown to facilitate shuttling of NF-
B from the nucleus to the cytoplasm, thereby reducing nuclear levels of I
B and terminating NF-
B-dependent gene transcription (2). The capacity of HT to delay reexpression of I
B-
might contribute, at least in part, to the sustained activation of NF-
B and expression of TNF-
. Because the promoter for I
B-
contains a binding site for NF-
B, the delay in NF-
B activation may contribute to the subsequent delay in I
B-
reexpression; however, the disproportionately greater effect of HT on the latter process suggests that HT may exert additional effects that reshape expression of NF-
B-dependent genes.
In the present study, we have shown that exposing THP-1 cells to 32°C or 28°C markedly enhances early activation of IKK- and IKK-
and modestly prolongs activation of each of these kinases. We used phosphospecific antibodies that recognize both IKK-
and -
, but only when phosphorylated on Ser180/181. IKK-
targets I
B-
for degradation (24), thereby promoting nuclear translocation of NF-
B, and in this study, the appearance of phosphorylated IKK-
temporally correlated with the loss of I
B-
and the nuclear translocation of NF-
B RelA/p65. While IKK-
appears less important than IKK-
for phosphorylating I
B-
, it plays a major role in the transactivation of NF-
B through phosphorylation of histone H3 (35), the NF-
B corepressor SMRT (19), and the transactivation domain of p65 at Ser536 (29). Considering these multiple actions of I
B kinase, prolonged IKK phosphorylation in hypothermic cells is likely to contribute to sustained NF-
B activation and TNF-
generation during exposure to HT.
LPS stimulates NF-B activation through bifurcating Toll-like receptor 4 (TLR4)-activated myeloid differentiation protein-88 (MyD88)-dependent and MyD88-independent, and TRIF [Toll/IL-1 receptor (TIR) domain-containing adaptor inducing IFN-
]-dependent pathways (1). The former stimulates early NF-
B activation, and the latter leads to delayed but prolonged NF-
B activation (17). The effect on IKK activation and p65 nuclear translocation in the present study suggests that HT either modifies both MyD88-dependent and -independent TLR4-activated pathways or exerts its effects on a final common pathway. We have previously shown that HT causes similar augmentation of TNF-
expression in response to the TLR4-independent agonists, opsonized zymosan, and toxic shock syndrome toxin-1 (13), suggesting that HT more likely modifies signaling events within a final common pathway leading to IKK-
and -
activation. We acknowledge that other transcription factors in addition to NF-
B regulate TNF-
transcription. While we have shown that HT augments activation of NF-
B without affecting activator protein-1 (13), these data do not exclude effects on additional transcription factors that might regulate the expression of proinflammatory cytokine genes.
In summary, we have shown that exposure to clinically relevant degrees of HT for as little as 2 h can increase the sensitivity to LPS and augment the magnitude and duration of TNF- expression in cultured human mononuclear cells. We have shown that these effects may be mediated in part through amplification of the final common pathway leading to activation of the pleiotropic transcription factor NF-
B. These studies indicate the complexities of predicting the effects of HT in intact animals and underscore the importance of elucidating the mechanisms though which HT exerts its biological effects.
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GRANTS |
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FOOTNOTES |
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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.
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