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(Chest. 2004;125:1483-1491.)
© 2004 American College of Chest Physicians

Hypothermia Induces Anti-Inflammatory Cytokines and Inhibits Nitric Oxide and Myeloperoxidase-Mediated Damage in the Hearts of Endotoxemic Rats*

Philip O. Scumpia, BS; Paul J. Sarcia, BS; Kindra M. Kelly, BS; Vincent G. DeMarco, PhD and Jeffrey W. Skimming, MD

* From the Departments of Pediatrics (Mr. Scumpia and Mr. Sarcia) and Physiology and Functional Genomics (Ms. Kelly), University of Florida, Gainesville, FL; and Department of Child Health (Dr. Skimming and Dr. DeMarco), University of Missouri, Columbia, MO.

Correspondence to: Jeffrey W. Skimming, MD, Department of Child Health, One Hospital Dr, Columbia, MO 65211; e-mail: skimmingj{at}missouri.edu


   Abstract
TOP
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
Study objectives: The impairment of cardiac contractility during endotoxemia involves induction of nitric oxide formation through a cascade of events initiated by overexpression of proinflammatory cytokines. We previously showed that hypothermia attenuates endotoxin-induced overexpression of nitric oxide in rat lungs. In the present study, we tested the hypothesis that hypothermia protects against endotoxin-induced myocardial inflammation by changing the balance of pro- and anti-inflammatory cytokines, inhibiting myeloperoxidase, an indicator of neutrophil activity, and inhibiting nitric oxide-mediated protein damage.

Design: Rats were randomized to treatment with either hypothermia (n = 6; 18 to 24°C) or normothermia (n = 6; 36 to 38°C). Endotoxin (15 mg/kg) was administered intravascularly to anesthetized animals, and heart tissue was harvested 150 min later.

Measurements and results: Using enzyme-linked immunosorbent assays (ELISAs), we found that hypothermia induced myocardial expression of the anti-inflammatory cytokines interleukin (IL)-4 and IL-10, while decreasing concentrations of the pro-inflammatory cytokines IL-1ß and growth-related oncogene/cytokine-induced neutrophil chemoattractant (rat homolog of IL-8). Electromobility shift assay revealed that hypothermia inhibited the nuclear translocation of nuclear factor-{kappa}B. Reverse transcriptase-polymerase chain reaction and Western blot assays revealed that hypothermia attenuated the endotoxin-induced overexpression of both inducible nitric oxide synthase (iNOS) messenger RNA and iNOS protein, respectively. Hypothermia also attenuated nitric oxide-mediated myocardial protein damage, as determined by a nitrotyrosine ELISA. Myocardial myeloperoxidase content, an indicator of neutrophil accumulation and oxidative activity, was also inhibited by hypothermia in endotoxemic rats.

Conclusion: These data demonstrate that hypothermia induces an anti-inflammatory cytokine profile, inhibits neutrophil aggregation, and inhibits the formation of nitric oxide during endotoxemia in the rat.

Key Words: growth-related oncogene/chemoattractant • inducible nitric oxide synthase • interleukin-4 • interleukin-10 • lipopolysaccharide • nitrotyrosine • nuclear factor-{kappa}B


   Introduction
TOP
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
Heart failure is characterized by myocardial depression and elevated proinflammatory cytokines.12 Animal studies34 suggest that elevated levels of the pro-inflammatory cytokine tumor necrosis factor (TNF)-{alpha} cause myocardial depression similar to that seen in experimental and clinical models of heart failure. In those patients with Gram-negative bacterial sepsis, the lipopolysaccharide (LPS) component of the bacterial membrane induces expression of various cytokines that act to depress myocardial function.5 Administration of LPS alone into healthy humans causes a sepsis-like syndrome involving myocardial dysfunction.6 Sepsis-induced expression of circulating cytokines, such as interleukin (IL)-1ß and TNF-{alpha}, accounts for some of the humorally mediated myocardial dysfunction.7 Endotoxin can cause local production of TNF-{alpha} and IL-8, which contributes to myocardial dysfunction and neutrophil-mediated myocardial damage.8910 Induction of both circulating and intramyocardial cytokines therefore contribute to myocardial depression during Gram-negative sepsis.

During sepsis, the balance between pro- and anti-inflammatory cytokines shifts in support of the proinflammatory milieu. The proinflammatory cytokines IL-1ß, TNF-{alpha}, and interferon-{gamma}, derived from T-helper type 1 cells, dominate the anti-inflammatory cytokines derived from T-helper type 2 cells, including IL-10 and IL-4. IL-10 has been shown to inhibit proinflammatory cytokine expression,111213 nuclear factor (NF)-{kappa}B activation,14 and nitric oxide production.15 Other benefits of IL-10 include promoting apoptosis in both neutrophils16 and monocytes.17 IL-4, however, has been shown to inhibit proinflammatory cytokine production by monocytes.171819 Increasing the concentrations of both IL-10 and IL-4 should therefore be expected to ameliorate the inflammatory consequences of sepsis, including myocardial dysfunction.

Hypothermia is often used as a means of preserving the heart during cardiopulmonary bypass and has also been used experimentally as means of inhibiting the inflammatory consequences of both severe brain trauma20 and bacterial meningitis.2122 In cultured T cells, hypothermia induces an anti-inflammatory cytokine profile after an inflammatory stimulus.23 Recently, we24 demonstrated that hypothermia inhibits the intrapulmonary expression of inducible nitric oxide synthase (iNOS) and the intrapulmonary formation of nitric oxide induced by endotoxin in rats. In this study, we examined the effects of hypothermia on intramyocardial pro- and anti-inflammatory cytokine expression and nitric oxide-mediated myocardial injury during endotoxemia. We also investigated whether hypothermia caused myocardial inhibition of NF-{kappa}B, a key transcriptional mediator of LPS-induced proinflammatory cytokine and iNOS expression.


   Methods and Materials
TOP
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 References
 
Surgical Preparation and Endotoxin Administration
Fifteen male Sprague-Dawley rats (Charles River Laboratory; Key Lois, FL) [280 to 350 g] were used for the experiments. All rats were fed a standard laboratory chow and were provided water ad libitum until the day of the experiment. The Institutional Animal Use and Care Committee of University of Florida approved of the experiments, and the care and handling of the animals were in accordance with National Institutes of Health guidelines. Animals were preanesthetized with brief exposure to halothane followed by IM injections of ketamine (100 mg/kg) and xylazine (10 mg/kg). Twelve rats were randomized into two groups: hypothermia (n = 6; 18 to 24°C) and normothermia (n = 6; 36 to 38°C). The three remaining rats were sham instrumented (36 to 38°C; no LPS). The rats in the hypothermia group were placed on an ice pack, while the rats in the normothermia and sham groups were placed on a heating pad. A rectal temperature probe was inserted and temperature was maintained within the temperature range for the group. Polyethylene-50 catheters were inserted in the right carotid artery for continuous BP monitoring using a polygraph (model MP 100; Biopac; Santa Barbara, CA) and fluid infusion. A tracheostomy was then performed, and a 14-gauge catheter was secured in the trachea and connected to a mechanical breathing circuit.

After securing the airway, all animals were paralyzed with IV pancuronium bromide (1 mg/kg). Ventilation was controlled with a small animal ventilator (Rodent Model 683; Harvard Apparatus; South Natick, MA), using a 4-mL tidal volume of room air at 35 breaths/min. Samples of arterial blood (70 µL) were analyzed using an iSTAT 200 portable blood gas analyzer (Abbott Laboratories; Abbott Park, IL). Before starting the experiments, the ventilation rate was adjusted to ensure a PaCO2 between 40 mm Hg and 45 mm Hg. The animals were allowed to acclimate to the experimental conditions for at least 20 min before collecting any data or administering endotoxin. Afterwards, a 15 mg/kg dose of Escherichia coli LPS (Sigma-Aldridge Corporation; St. Louis, MO) was administered intravascularly to induce endotoxemia in the normothermic and hypothermic groups. Animals were monitored for 150 min after the infusion of endotoxin or saline solution.

Tissue Sample Collection
The animals were killed with an overdose of saturated KCl. The heart was quickly removed, cut into four pieces, and each piece was snap frozen in liquid nitrogen and stored at – 80°C for subsequent analysis.

Cytokine, Chemokine, and Nitrotyrosine Assays
IL-1ß, IL-4, and IL-10 concentrations from supernatants derived from lung tissue homogenates were measured using Endogen Rat Interleukin ELISA kits (Pierce Endogen; Rockford, IL) and expressed as nanograms per gram wet weight (gww). Growth-related oncogene/cytokine-induced neutrophil chemoattractant (GRO/CINC-1) concentrations were measured using the TiterZyme-EIA rat GRO/CINC-1 Enzyme Immunometric Assay Kit (Assay Designs; Ann Arbor, MI). Absorbencies were determined at 450 nm using a Powerwave X microplate reader (Biotek Instruments; Winooski, VT), and concentrations were calculated using the equation derived from a linear standard curves for each cytokine or chemokine. The nitrotyrosine concentration of sample extracts was determined using an enzyme-linked immunosorbent assay (ELISA) kit for nitrotyrosine (Cayman Chemicals; Ann Arbor, MI) according to the protocol of the manufacturer.

Nuclear Protein Extraction
Nuclear protein extracts were prepared from heart tissue homogenized in 2 mL of phosphate-buffered saline solution. All nuclear extraction procedures were performed on ice with chilled reagents using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Endogen) according to the instructions of the manufacturer. The final supernatant derived from the nuclear pellet was placed in a clean, prechilled tube and stored at – 80°C until electromobility shift assay (EMSA) or Western blot was done on the nuclear protein extracts.

EMSA for NF-{kappa}B
The NF-{kappa}B oligonucleotide probe (Promega Corporation; Madison, WI) [5'-AGT TGA GGG GAC TTT CCC AGG C-3'] was labeled with [{gamma}-32P]adenosine triphosphate using T4 polynucleotide kinase (Life Technologies; Invitrogen; Carlsbad, CA) and purified in push columns (Stratagene; Cedar Creek, TX). For EMSA, 10 µg of nuclear proteins were preincubated with EMSA buffer (12 mmol/L hydroxylethyl piparazine-ethanesulfonic acid pH 7.9, 4 mmol/L Tris-HCl pH 7.9, 25 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L ethylenediamine tetra-acetic acid, 1 mmol/L dithiothreitol, 50 ng/mL poly[d(I-C)], 12% glycerol volume/volume, and 0.2 mmol/L phenylmethyl-sulfonylfluoride) on ice for 10 min before addition of the radiolabeled probe for an additional 10 min. Protein-nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29:1 ratio of acrylamide:bisacrylamide) and run in 0.5x TBE (45 mmol/L Tris-HCl, 45 mmol/L boric acid, 1 mmol/L ethylenediamine tetra-acetic acid) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3 M paper (Whatman; Clifton, NJ), dried under a vacuum at 80°C for 1 h, and exposed to photographic film at – 80°C.

Reverse Transcriptase-Polymerase Chain Reaction
Reverse Transcription: Total RNA was extracted from snap-frozen heart samples using TRIzol Reagent (Life Technologies). The integrity of isolated total RNA was determined using 1% agarose gel electrophoresis and RNA concentrations were determined by ultraviolet light absorbance at a wavelength of 260 nm. RNA samples were incubated with ribonuclease-free deoxyribonuclease 1 (Amersham Pharmacia Biotech; Piscataway, NJ) for 15 min at 37°C and extracted by a phenol-chloroform technique. Moloney murine leukemia virus reverse transcriptase and random hexamer primers (Ready- to-Go RT-PCR Beads; Amersham Pharmacia Biotech) were used to reverse transcribe all messenger RNA species to complimentary DNA. The reaction incubated for 30 min at 42°C in a PTC-200 DNA Engine thermocycler (MJ Research; Watertown, MA). The complementary DNA samples were then incubated at 95°C for 5 min in the thermocycler to inactivate the reverse transcriptase. Samples were screened for genomic DNA contamination by carrying samples through the polymerase chain reaction (PCR) procedure without adding reverse transcriptase.

PCR: Reverse transcriptase-generated complementary DNA encoding iNOS and ß-actin were amplified using PCR. ß-actin, a housekeeping gene, was used as an internal standard. The oligonucleotide primer sequences (Table 1 ) were designed in accordance with published rat DNA sequences for iNOS (accession No. D14051)25 and ß-actin (accession No. V01217 and No. J00691).26


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  		Table 1.. Oligonucleotide Primers for Reverse Transcriptase-PCR

 
The experimental conditions for iNOS, and ß-actin PCR reactions were as follows: initial denaturation at 95°C for 5 min followed by 32 cycles of amplification at 94°C for 1 min and 72°C for 1.5 min. A negative control for each set of PCR reactions contained water instead of the DNA template. All PCR products (8 µL) were electrophoretically separated on a 1% agarose gel and then stained with ethidium bromide. A Gel Doc 2000 Gel Documentation System (Bio-Rad Laboratories; Hercules, CA) was used to visualize the PCR products. Densitometric techniques were then performed to quantify the DNA band densities using NIH software (Scion Corporation; Frederick, MD).

Immunoblotting Assay for iNOS and p65
For the detection of iNOS protein in whole-cell lysates, frozen heart samples were thawed immediately before analysis and homogenized in five volumes of boiling lysis buffer (1% sodium dodecyl sulfate [SDS], 1.0 mmol/L sodium orthovanadate, and 10 mmol/L Tris pH 7.4). After microwaving for 10 to 15 s, the crude homogenates were centrifuged at 15°C for 5 min at 16,000g, and the supernatants were collected for analysis. The protein concentration of each sample was measured using a BCA protein assay kit (Pierce Chemical). An equal volume of 2x sample buffer (250 mmol/L Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% b-mercaptoethanol) was added to all the samples and boiled for 3 to 5 min.

Proteins were separated by SDS-gel electrophoresis. Equal amounts of protein (65 µg) were loaded onto each well of 7.5% Tris-glycine precast polyacrylamide gels (Bio-Rad Laboratories) and separated by gel electrophoresis at 50 V of constant current for 180 min using a Mini-Protean electrophoresis system (Bio-Rad Laboratories). Lysate from cytokine-activated murine macrophages was used as a positive control. Then the proteins were transferred from gels to nitrocellulose membranes (Bio-Rad Technologies) at 100-V constant current for 60 min in transfer buffer (25 mM Tris, 190 mmol/L glycine, 20% methanol, 0.05% SDS). The nitrocellulose membranes were then immediately placed into blocking buffer (5% nonfat dry milk, 10 mmol/L Tris pH 7.5, 100 mmol/L sodium chloride, 0.1% Tween-20) and left at room temperature for 60 min. After blocking, the membranes were incubated overnight at 4°C in primary antibody solution (1:1000 dilution in blocking buffer, murine monoclonal iNOS, IgG2a, antibody; Transduction Laboratories; Lexington, KY). Horseradish peroxidase conjugated sheep anti-mouse IgG antibody (1:2000 dilution in blocking buffer; Amersham Pharmacia Biotech) was used as a secondary antibody. Bound antibody was detected by chemiluminescence (ECL plus kit; Amersham Pharmacia Biotech). The bands were expected at a size of 130 kd for iNOS.

For the detection of the p65 subunit of NF-{kappa}B in the nucleus, nuclear extracts were also subjected to the Western blot technique using a rabbit polyclonal anti-p65 primary antibody (1:500 dilution in blocking buffer; Santa Cruz Biotechnology; Santa Cruz, CA) and horseradish peroxidase conjugated anti-rabbit antibody (1:2000 dilution in blocking buffer; Amersham Pharmacia Biotech). Densitometric techniques were performed to quantify the protein band densities.

Myeloperoxidase Assay
Myeloperoxidase activity was used to assess neutrophil accumulation in the heart tissue using a previously reported method.27 Briefly, thawed heart samples were weighed and homogenized on ice in 0.01 mol/L KH2PO4 at a ratio of 1 volume tissue to 15 volumes of buffer. After centrifugation at 10,000g for 20 min at 4°, the pellets were resuspended by sonication in cetyltrimethylammonium bromide buffer (13.7 mmol cetyltrimethylammonium bromide, 50 mmol/L KH2PO4, 50 mmol/L acetic acid; pH 6.0) at a ratio of 1 to 5 weight to volume. The supernatant was kept for ELISA analysis (see previous text). The suspension was centrifuged again at 10,000g for 15 min, and the pellet was discarded. The supernatant was then incubated in a 60°C water bath for 2 h. Myeloperoxidase activity of the supernatant was measured by the H2O2-dependent oxidation of tetramethylbenzidine. Absorbance was determined at 650 nm and compared with a linear standard curve.

Statistical Analysis
Differences in the means among two or three treatment groups were detected using t tests or one-way analysis of variance, respectively. Post hoc analyses using Student-Newman-Keuls test were performed if the analysis of variance revealed an effect of treatment. A significance level was set as 0.05. Data analyses were performed using SigmaStat for Windows, Version 2.03 (SPSS; Chicago, IL). Data are reported in the text and figures as means ± SE.


   Results
TOP
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
Effects of Hypothermia on Cytokine Expression
The concentration of IL-4 in the hearts of hypothermia-treated rats (1,825 ± 115 ng/gww) was more than threefold greater than that found in the normothermia (574 ± 196 ng/gww, p < 0.001) and sham (447 ± 83 ng/gww, p < 0.001) groups (Fig 1 , top left, A). Similarly, IL-10 expression was induced by hypothermia (2,439 ± 185 pg/gww) compared to the normothermia (764 ± 129 pg/gww, p < 0.001) and sham (442 ± 79 pg/gww; p < 0.001) groups (Fig 1, top right, B).



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  		Figure 1.. ELISAs were performed to determine IL-4 (top left, A), IL-10 (top right, B), IL-1ß (bottom left, C), and GRO-CINC-1 (bottom right, D) concentrations in protein extracts from rat hearts. Densitometry performed on separate immunoblots indicates that IL-4 and IL-10 increase and IL-B and GRO/CINC-1 decrease in response to hypothermia compared to normothermia (p < 0.05 for all). Capped bars indicate the mean ± SE of relative protein density. *Different (p < 0.05) from the normothermia group as determined by paired comparisons. Sample sizes are 3, 6, and 6 for the sham, normothermia, and hypothermia groups, respectively.

 
The concentration of IL-1ß in the hearts of normothermia-treated rats (3.2 ± 0.2 ng/gww) was over threefold greater than that found in the hypothermia group (1.0 ± 0.1 ng/gww; p < 0.001; Fig 1, bottom left, C). Similarly, LPS caused an increase in GRO/CINC-1 expression in the normothermia group (38 ± 5 ng/gww) compared to the control group (0.2 ± 0.1 ng/gww, p < 0.001; Fig 1, bottom right, D), and that increase was almost completely inhibited by hypothermia (0.63 ± 0.2 ng/gww, p < 0.001).

Effects of Hypothermia on NF-{kappa}B Activation and Nuclear Translocation
Results from the EMSA (Fig 2 , top, A) confirm that hypothermia inhibits NF-{kappa}B activation and translocation into the nuclei of myocardial cells compared to normothermia. Densitometric analysis indicates there is a 35-fold increase in nuclear level of NF-{kappa}B (175 ± 10 U) compared to the hypothermia group (5 ± 4 U; p < 0.001). Similarly, a densitometric analysis of an immunoblot using an antibody to p65 (Fig 2, bottom, B), the active subunit of NF-{kappa}B, revealed that hypothermia inhibited the level of NF-{kappa}B (117 ± 10 U) in nuclear extracts as compared to the normothermia group (61 ± 13 U; p = 0.007).



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  		Figure 2.. Top, A: The effect of hypothermia on NF-{kappa}B DNA binding activity in rat heart homogenates after treatment with LPS. Nuclei were extracted from tissue homogenates and subjected to EMSA. The radiograph and the underlying graph indicate that hypothermia blunts the activation of NF-{kappa}B compared to normothermia (p < 0.001). Quantification of the radiographic signal was determined by standard densitometry. Bottom, B: Western immunoblot analysis of nuclear protein extracts from rat heart using an antibody to p65 show reduced levels of this NF-kB protein subunit in the nucleus, suggesting that hypothermia reduces LPS-induced activation of NF-{kappa}B (p < 0.01). This result is consistent with the result from the gel shift assay. *Hypothermia group is different (p < 0.05) from the normothermia group as determined by paired comparisons. Samples sizes are 5 and 6 for the normothermia and hypothermia groups, respectively.

 
Effects of Hypothermia on iNOS Expression
The relative density of iNOS messenger RNA normalized to ß-actin in rat myocardium was lower in the hypothermia group than the normothermia group (p = 0.001; Fig 3 , top, A). Densitometric analysis of iNOS protein indicated higher levels of the protein in the myocardium of the normothermia group than in the hypothermia group (p = 0.013).



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  		Figure 3.. Top, A: Analysis of rat heart messenger RNA extracts using conventional reverse transcriptase-PCR to detect iNOS messenger RNA. ß-actin was used as an internal control for the amount of iNOS complementary DNA present in each sample (expressed as relative density). Hypothermia inhibits the endotoxin-induced increase in iNOS messenger RNA (p < 0.001). Bottom, B: Western immunoblot of protein extracts from rat heart homogenates visualized using monoclonal anti-iNOS antibody. Densities of iNOS protein bands in rat hearts were lower in the hypothermia group than in the normothermia group (p = 0.013). Capped bars indicate the mean treatment concentration ± SE determined by densitometry. *Hypothermia group is different from the normothermia group (p < 0.05). Samples sizes are six each for the normothermia and hypothermia groups, respectively. bp = base-pair.

 
Effects of Hypothermia on Nitrotyrosine Formation
The formation of nitrotyrosine was induced by endotoxin in the normothermia group compared to the sham group (32 ± 5 pg/gww vs 11 ± 2 pg/gww, p < 0.05; Fig 4 , top, A). Hypothermia (16 ± 1 pg/gww) attenuated the endotoxin-induced increase in nitrotyrosine formation in the heart tissue (p < 0.05).



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  		Figure 4.. Top, A: ELISA using a monoclonal antibody revealed that hypothermia attenuated the effect of endotoxin on nitrotyrosine concentrations in protein extracts of rat heart (p < 0.001). Bottom, B: Myeloperoxidase content assay performed on extracts from rat heart demonstrates that myeloperoxidase content is decreased in the hypothermia group (p < 0.001) compared to the normothermia group. Myeloperoxidase content of the hypothermia group is no different from baseline level seen in the sham-treated group (p > 0.05). Capped bars indicate the mean treatment concentration ± SE. *Different (p < 0.05) from the normothermia group as determined by paired comparisons. Samples sizes are 3, 6, and 6 for the sham, normothermia, and hypothermia groups, respectively.

 
Effects of Hypothermia on Myeloperoxidase
The normothermia group exhibited a 70-fold increase (p < 0.003) in heart myeloperoxidase concentration (1.43 ± 0.24 U/gww) compared to the sham group (0.02 ± 0.0004 U/gww; Fig 4, bottom, B). Hypothermia (0.15 ± 0.11 U/gww) attenuated the LPS-induced increase in myeloperoxidase concentration (p < 0.001) and was no different from the sham group (p > 0.05).


   Discussion
TOP
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
The anti-inflammatory benefits of hypothermia have been suggested to result from a generalized temperature-dependent decrease in metabolism.28 However, our results suggest that the anti-inflammatory effects of hypothermia involve increased expression of some anti-inflammatory mediators. Further studies are needed which explore the use of IL-4 and IL-10 as agents that offer the therapeutic advantages of yet avoid the potential hazards of hypothermia therapy such as those reported with rewarming.2930

The morbidity and mortality associated with sepsis has been linked to endotoxin induction of proinflammatory cytokines, most notably IL-1ß and IL-8. In our study, hypothermia inhibited the expression of these two cytokines but induced the expression of two anti-inflammatory cytokines, IL-4 and IL-10. It is likely that hypothermia stimulates T-helper type 2 cells to produce an anti-inflammatory cytokine profile.23 Additionally, there is abundant evidence that IL-10 inhibits many aspects of endotoxin-mediated inflammation.14151631 Although the mechanisms responsible for IL-10 and IL-4 anti-inflammatory effects are poorly understood, recent evidence suggests that IL-10 induces macrophages to express heme oxygenase-1, a stress-inducible protein with potential anti-inflammatory effect, via a p38 mitogen-activated protein kinase-dependent pathway.32 Thus, we speculate that the increase in the levels of IL-10 and IL-4 in the hearts of hypothermic rats inhibits the inflammation caused by endotoxin seen in our experiment.

The activation of the key inflammatory mediator NF-{kappa}B has also been shown to be important in immunologic reactions resulting from endotoxemia such as cytokine3334 and nitric oxide3536 production. Others have shown that NF-{kappa}B inhibition may have beneficial effects during endotoxemia.3738 It is likely that the decreased activation of NF-{kappa}B observed in the heart of hypothermia rats is responsible for the decreased expression of cardiac IL-1ß, GRO/CINC-1, and iNOS.

The overproduction of nitric oxide by iNOS has also been shown to be a major contributor to the pathogenesis of endotoxemia. Inhibition of iNOS activity or expression with various compounds during endotoxemia has been shown to improve cardiac function,39 inhibit organ injury40 and shock,41 and improve survival42 in rodent models. In a previous study,24 we found that hypothermia inhibits endotoxin-induced intrapulmonary nitric oxide formation and regulates iNOS at the transcriptional level. We also found that hypothermia attenuated nitric oxide-mediated protein damage in the lungs as evidenced by decreased nitrotyrosine concentrations in the lung tissue.24 Consistent with those data, we found that hypothermia attenuated the endotoxin-induced increase in iNOS messenger RNA and protein in heart tissue as well as the increased concentration of nitrotyrosine residues in the heart proteins of the endotoxin-treated rats. Thus, our data suggest that hypothermia protects against cardiopulmonary nitric oxide-related organ injury caused by endotoxin, perhaps by inhibition of locally formed nitric oxide.

Endotoxemia increases circulating levels of the neutrophil chemoattractant IL-8,10 and also up-regulates the neutrophil chemoattractant GRO/CINC-1 (human IL-8 homolog) in rat cardiac myocytes.43 In whole animals, endotoxin-induced accumulation of neutrophils in various organs can lead to multiple organ failure. Expression of myeloperoxidase, an enzyme involved in neutrophil oxidative bursts and a marker of neutrophil-mediated injury, is increased in the myocardium during endotoxemia.404244 In our experiment, hypothermia decreased the concentration of GRO/CINC-1 and myeloperoxidase in rat hearts. These results suggest that hypothermia decreases both myocardial neutrophil accumulation and activity during endotoxemia.

This study may offer some insight into the cardioprotective effects of hypothermia that may translate to other systemic inflammatory disorders, such as cardiopulmonary bypass, during which hypothermia is often used. Similarities exist in the pathology of endotoxemia to that of surgical procedures involving cardiopulmonary bypass. Proinflammatory cytokine release, NF-{kappa}B activation, iNOS expression, and neutrophil accumulation occur during the ischemic conditions of cardiopulmonary bypass as well. A previous study45 showed that hypothermic cardiopulmonary bypass results in a greater expression of IL-10 and better myocardial protection than normothermic cardiopulmonary bypass in piglets. Our data are consistent with those findings.

Several limitations to this study exist. One important limitation of this study is that rewarming the animals was not attempted. However, we prospectively designed this study to elucidate the effects of hypothermia on the parameters tested rather than study the rationale for hypothermia therapy. Interspecies differences between rats and humans do exist and are highlighted by the finding that our rats continued to have a normal cardiac rhythm at a core temperature of 18 to 24°C. At these temperatures, human subjects ordinarily spontaneously arrest their cardiac function. Another limitation of our study is the choice of endotoxin administration as our model for sepsis. Although endotoxin administration is a well-established protocol for systemic inflammatory responses, it does not exactly mimic a bacterial infection or the conditions associated with cardiopulmonary bypass. We chose endotoxemia as our model because of the simplicity of the study design and the ease to which we can test the anti-inflammatory properties of hypothermia in rodents.

In summary, hypothermia results in an induction of a T-helper type 2 response involving increased expression of IL-10 and IL-4 in the heart of endotoxemic rats. On the contrary, myocardial expression of the inflammatory cytokines IL-1ß and GRO/CINC-1 was inhibited. Hypothermia also inhibited myocardial expression of nuclear NF-{kappa}B, iNOS, nitrotyrosine, and myeloperoxidase. These data are consistent with recent findings from our laboratory that hypothermia stimulates IL-10 production and reduces the proinflammatory response to endotoxin in the lungs of rats.46 Whether IL-4 and/or IL-10 administration can mimic the therapeutic effects yet avoid the pathologic consequences of hypothermia warrants further investigation.


   Footnotes
 
Abbreviations: ELISA = enzyme-linked immunosorbent assay; EMSA = electromobility shift assay; GRO/CINC-1 = growth-related oncogene/cytokine-induced neutrophil chemoattractant; gww = gram wet weight; IL = interleukin; iNOS = inducible nitric oxide synthase; LPS = lipopolysaccharide; NF = nuclear factor; PCR = polymerase chain reaction; SDS = sodium dodecyl sulfate; TNF = tumor necrosis factor

This work was supported in part by grants from the National Institutes of Health (5M01RR000082-390655) and American Heart Association (0151064B) awarded to Dr. Skimming.

Received for publication April 21, 2003. Accepted for publication September 17, 2003.


   References
TOP
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 References
 

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