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Am J Physiol Heart Circ Physiol 285: H2500-H2509, 2003. First published August 28, 2003; doi:10.1152/ajpheart.00432.2003
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Macrophage migration inhibitory factor is a cardiac-derived myocardial depressant factor

Leslie B. Garner,1,* Monte S. Willis,2,3,* Deborah L. Carlson,1,2 J. Michael DiMaio,4 Michael D. White,4 D. Jean White,2 Glenn A. Adams, IV,4 Jureta W. Horton,2 and Brett P. Giroir1

1Department of Pediatrics, University of Texas Southwestern, Dallas 75390-9063; 2Department of Surgery, University of Texas Southwestern, Dallas 75390-9160; 3Department of Pathology, University of Texas Southwestern, Dallas 75390-9072; and 4Department of Cardiothoracic Surgery, University of Texas Southwestern, Dallas, Texas 75390-9130

Submitted 8 May 2003 ; accepted in final form 20 August 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Macrophage migration inhibitory factor (MIF) is a pluripotent proinflammatory cytokine that is ubiquitously expressed in organs, including the heart. However, no specific role for MIF in modulating cardiac performance has yet been described. Therefore, we examined cardiac MIF expression in mice after LPS challenge (4 mg/kg) and tested the hypothesis that MIF is a mediator of LPS-induced cardiac dysfunction. Western blots of whole heart lysates, as well as immunohistochemistry, documented constitutive MIF protein expression in the heart. Cardiac MIF protein levels significantly decreased after LPS challenge, reaching a nadir at 12 h, and then returned to baseline by 24 h. This pattern was consistent with MIF release from cytoplasmic stores after endotoxin challenge. After release of protein, MIF mRNA levels increased 24–48 h postchallenge. To determine the functional consequences of MIF release, we treated LPS-challenged mice with anti-MIF neutralizing antibodies or isotype control antibodies. Anti-MIF-treated animals had significantly improved cardiac function, as evidenced by a significant improvement in left ventricular (LV) fractional shortening percentage at 8, 12, 24, and 48 h after endotoxin challenge. In support of these findings, perfusion of isolated beating mouse hearts (Langendorff preparation) with recombinant MIF (20 ng/ml) led to a significant decrease in both systolic and diastolic performance [LV pressure (LVP), positive and negative first derivative of LVP with respect to time, and rate of LVP rise at developed pressure of 40 mmHg]. This study demonstrates that MIF mediates LPS-induced cardiac dysfunction and suggests that MIF should be considered a pharmacological target for the treatment of cardiac dysfunction in sepsis and potentially other cardiac diseases.

cardiac dysfunction; innate immunity; sepsis; lipopolysaccharide

MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) is a pluripotent cytokine whose mechanisms of action remain elusive despite nearly four decades of study. Although crystallized as a trimer, its physiologically relevant oligomerization state remains unclear, its putative membrane receptor is unknown, and the physiological relevance of its intracellular enzymatic activity as a tautomerase and oxidoreductase remains uncertain. Despite our lack of a precise mechanistic understanding, many studies have demonstrated that MIF has an important role in such diverse diseases as rheumatoid arthritis, delayed-type hypersensitivity, inflammatory lung disease, cancer, myocardial infarction, and, perhaps most importantly, septic shock (26). During septic shock, MIF is increased in the plasma of animals and humans, and the blockade of MIF activity by monoclonal or polyclonal antibodies results in a marked improvement in the survival of animals with experimentally induced sepsis (7, 11). However, the pathophysiological mechanism(s) for this survival benefit remains uncertain.

We have investigated the possibility that MIF is an inducer of myocardial dysfunction that is known to contribute significantly to the morbidity and mortality of sepsis in humans (12, 22). In both human patients and animal models, sepsis-associated cardiac dysfunction is characterized by biventricular dilatation, decreased systolic contractility, and diminished diastolic relaxation (31, 34, 41). Most available data suggest that its pathogenesis is multifactorial with systemic and myocardial-derived cytokines such as TNF-{alpha} being necessary and sufficient to induce its onset (8, 21, 22).

In addition to TNF-{alpha}, we sought to identify other cardiac-derived proteins that might mediate, by paracrine or autocrine mechanisms, myocardial dysfunction in sepsis and potentially other cardiac diseases. Screening microarray analysis of cardiac gene expression in mice suggested that MIF was expressed in the heart and was differentially regulated by LPS (unpublished data). Moreover, MIF has been shown to be constitutively expressed in the heart and upregulated in a model of acute myocardial infarction in rats (45). Given the data that MIF inhibition improves outcome in animals with experimental sepsis and the fact that MIF can be upregulated in the heart experimentally, we designed these studies to determine the expression pattern of MIF in cardiomyocytes in vivo, to examine whether this expression was altered by LPS challenge, and, most importantly, to evaluate MIF's potential physiological effect on cardiac function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Antibodies and cytokines. Polyclonal goat anti-human MIF IgG and recombinant human MIF (rMIF; R&D Systems, Minneapolis, MN) were reconstituted in PBS and 0.1% BSA in PBS, respectively, measured into aliquots, and stored at –20°C until use. This polyclonal goat anti-human MIF antibody has been shown to cross-react with murine MIF and was used in Western blot experiments (3). A polyclonal rabbit anti-goat IgG-horseradish peroxidase (HRP) (Bio-Rad, Hercules, CA) used as a secondary antibody for Western blots was stored at 4°C until use. A polyclonal rabbit anti-rat MIF IgG (Torrey Pines BioLabs, Houston, TX) was used for immunohistochemistry and was shown to cross-react with murine MIF in Western blot experiments in our laboratory (data not shown). Two monoclonal mouse anti-mouse MIF IgG1 antibodies (XIV.15.5 and III.D.9, gift from Cytokine PharmaSciences) and a monoclonal mouse IgG1 isotype control antibody (HB-49, gift from Cytokine PharmaSciences) were used in the echocardiographic studies. These antibodies were raised after immunization with both mouse and human MIF and recognize both human and mouse MIF (personal communication, Dr. V. de la Cruz, Cytokine PharmaSciences). Previous studies have demonstrated in vivo neutralization of MIF activity by both the XIV.15.5 antibody (15, 29) and III.D.9 antibody (6, 13, 24).

Animals and experimental design. C57BL/6J mice (Jackson Labs, Bar Harbor, ME) and C3H/HeJ mice (Harlan, Indianapolis, IN) were obtained at 6–10 wk of age and maintained in a specific pathogen-free environment. Commercial chow and tap water were made available ad libitum. All animal protocols were reviewed and approved by the University of Texas Southwestern Medical Center Institutional Animal Care Advisory Committee and were in compliance with the rules governing animal use as published by the National Institutes of Health. C57BL/6J mice were injected intraperitoneally with 4 mg/kg Esherichia coli 0111:B4 LPS (Sigma-Aldrich, St. Louis, MO) and killed postinjection at time points indicated in the figures by CO2 asphyxiation and subsequent cervical dislocation. Uninjected mice were used as controls. Both anti-MIF antibodies (III.D.9 and XIV.15.5) and an isotypic control (HB-49) were injected (100 µg in 200 µl PBS) intraperitoneally 90 min before the LPS challenge in the echocardiogram studies. Whole hearts were removed, snap frozen in liquid nitrogen, and stored at –80°Cor fixed in 10% neutral-buffered formalin for 24 h and placed in 70% ethanol for immunohistochemistry. Whole blood was collected by retro-orbital bleeding using a small-diameter heparin-coated (to prevent local clotting in the capillary tube) microhematocrit capillary tube (Allegiance Healthcare, McGaw Park, IL), which immediately directed the blood from the eye into a serum separator tube containing clot activators (Vacutainer SST Gel & Clot activator tubes, Becton Dickinson, Franklin Lakes, NJ). The tube was inverted five times, allowed to clot for a minimum of 30 min, and centrifuged at 1,200 g for 10 min at 4°C. Care was taken to avoid hemolysis during collection of serum. Serum was transferred to a sterile snap-top tube and frozen at –80°C until assayed by ELISA.

Protein extraction and Western blotting. Hearts were thawed and homogenized on ice in Tris-buffered saline (TBS, 50 mM Tris and 150 mM NaCl, pH 7.5) containing 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 2 mM EDTA, and 1 mM PMSF. Lysate protein concentration was quantified using the Bio-Rad Protein Assay. Protein (20 µg) was diluted 2 parts sample with 1 part Laemmli sample buffer (Bio-Rad) and resolved on an 18% SDS polyacrylamide gel under reducing conditions. Prestained SDS-PAGE standards (Kaleidoscope Broad range, Bio-Rad Laboratories) were run with each gel to determine the approximate molecular weight of the detected bands. Additionally, rMIF (R&D Systems) was used as a positive control on initial Western blots. The gel was transferred to polyvinylidene difluoride membranes (NEN, Boston, MA) using a semidry transfer apparatus (Bio-Rad) at 15 V for 15 min. The membrane was then blocked with TBS-0.1% Tween 20 (TBS T) with 0.5% nonfat dry milk (Bio-Rad) for 30 min and incubated with polyclonal goat anti-human MIF IgG (1:750) in TBS-T with 0.5% nonfat milk overnight at 4°C. The membrane was washed three times for 10 min in TBS-T, incubated with rabbit anti-goat IgG-HRP (1:1,000) for 1 h at room temperature, and washed four times for 10 min with TBS-T. The membrane was then exposed to 5 ml of a mixture of luminol plus hydrogen peroxide under alkaline conditions (SuperSignal West Pico, Pierce, Rockford, IL) for 5 min, and the resulting chemiluminescent reaction was detected by Kodak X-OMAT AR Film (Eastman Kodak, Rochester, NY).

The quantification of the single band density with the approximate molecular mass of MIF (12.5 kDa) was determined using Quantity One software (Bio-Rad, version 4.4.0, build 36) after conversion of radiographic film to TIFF files (8-bit grayscale) using a Scanjet 7400c (Hewlett-Packard, Palo Alto, CA) and reported in arbitrary units (AU) per square millimeter. Densitometry was performed by outlining the MIF bands with the volume rectangle tool initially set on the control band of interest. This rectangle was then copied and pasted onto other bands that were completely outlined, and the volume analysis report was run.

RNA extraction, probe preparation, and Northern blotting. Hearts were thawed on ice, and total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol and quantified by spectrophotometry. An MIF-specific Northern probe was prepared by isolating DNA (DNeasy Tissue Kit, Qiagen, Valencia, CA) from an MIF plasmid (Research Genetics, Huntsville, AL) and subsequently cutting it with EcoRI and NotI restriction enzymes (Fisher Scientific, Pittsburgh, PA). The resultant DNA was resolved on a 1.2% agarose gel, purified (GenElute Agarose Spin Columns, Supelco, Bellefonte, PA), labeled with 5 µl [32P]dCTP (3,000 Ci/mmol) (Perkin-Elmer, Boston, MA) using Ready-To-Go Labeling Beads (Amersham Pharmacia, Piscataway, NJ), and purified in ProbeQuant Microcolumns (Amersham Pharmacia) according to manufacturers' protocols.

RNA (10 µg) was resolved on 1.2% agarose gels at 100 V for 1 h and transferred to a Hybond-N+ membrane (Amersham Pharmacia, Buckingham, UK) at 1.5 A for 1 h on a transfer electrophoresis unit (TransPhor PowerLid, Hoefer Scientific Instruments, San Francisco, CA). RNA was linked to the membrane for ~2 min using a GS Gene Linker set at C3 (Bio-Rad). The membrane was prehybridized in a hybridization oven (Sorvall Life Science, Greensboro, NC) in Perfect-Hyb Plus (Sigma, St. Louis, MO) for 1 h at 68°C. Sheared, denatured salmon testis DNA (100 µg/ml) was then added for 1 h, followed by the addition of ~0.1 µg probe labeled at >5 x 108 counts·min–1·µg–1. The blot was then hybridized for 12hat68°C in the hybridization oven followed by washing at 68°C in 2x standard saline citrate and 0.1% SDS. The membrane was washed for 1 h, the buffer was exchanged, and then the membrane was washed for an additional 1 h at 68°C. The membrane was wrapped in Saran Wrap, and mRNA was detected by Kodak X-OMAT AR film after 96 h (Eastman Kodak). The same membrane was stripped and then reprobed in a similar manner with radiolabeled {beta}-actin (0.1 µg probe labeled at >5 x 108 counts·min–1·µg–1) (Ambion, Austin, TX). Densitometry was performed as described above for the Western blots. The {beta}-actin mRNA bands served as a control against which to normalize the MIF mRNA densitometry.

Immunohistochemistry. Tissue was fixed in neutral buffered formalin, processed to paraffin, and subsequently immunostained at room temperature on a BioTek Solutions Techmate 1,000 automated immunostainer (Ventana Medical Systems, Tucson, AZ) using the Ultra-streptavidin biotin system with HRP and diaminobenzidine (DAB) chromogen (Signet Laboratories, Dedham, MA). Optimum primary antibody concentrations were predetermined using known positive control tissues (LPS-challenged rat as previously described) (4). Paraffin sections were cut at 3 µm on a rotary microtome, mounted on positively charged glass slides (POP100 capillary gap slides, Ventana Medical Systems), and air-dried overnight. Sections were then deparaffinized in xylene and ethanol, quenched with fresh 3% hydrogen peroxide for 10 min to inhibit endogenous tissue peroxidase activity, and rinsed with deionized water. Sections were incubated in unlabeled blocking serum for 15 min to block nonspecific binding of the secondary antibody and then incubated for 25 min with either the polyclonal rabbit anti-rat MIF IgG (1:400, Torrey Pines BioLabs, Houston, TX) diluted in 1% citrate buffer (BioPath, Oklahoma City, OK) or with buffer alone as a negative reagent control. A negative reagent control was run for each time point and for each organ. After washes in buffer, sections were incubated for 25 min with a biotinylated polyvalent secondary antibody solution (containing goat anti-rabbit IgG). Next, sections were washed with buffer, incubated in HRP-conjugated streptavidin-biotin complex for 15 min, washed again in buffer, and then incubated with two changes, 5 min each, of a freshly prepared mixture of DAB and H2O2 in buffer, followed by washing in buffer and then water. Sections were then counterstained with hematoxylin, dehydrated in a graded series of ethanol and xylene, and placed under coverslips. Slides were reviewed by light microscopy, and positive reactions with DAB were identified as a dark brown reaction product.

Determination of cardiac function in response to recombinant MIF. C57BL/6J and C3H/HeJ mice were used in the Langendorff assays as previously described (44). Briefly, 200 units of heparin sulfate were given intraperitoneally, the mice were killed 20 min later, and the heart was immediately removed and placed on ice in Krebs-Henseleit buffer [in mM: 2 NaHCO3, 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 11.1 glucose, pH 7.4, which was prepared fresh with demineralized, deionized water and bubbled with 95% O2-5% CO2 (PO2 590 mmHg, PCO2 38 mmHg)]. The aorta was cannulated with PE-50 tubing, and the heart was perfused in a retrograde manner through the aortic root with prefiltered, oxygenated Krebs-Henseleit buffer at a constant flow rate of 1.5 ml/min (temperature 37°C) and a recirculating volume of 100 ml. The heart was placed in a water-jacketed chamber to maintain constant temperature and humidity. PE-50 intramedic polyethylene tubing, connected to a Statham pressure transducer, was inserted into the left ventricle (LV) to measure LV pressure (LVP). Temperature was monitored using a 27-gauge thermistor needle inserted into the LV muscle. After instrumentation, hearts were allowed to stabilize for 10 min, and hearts that failed to achieve a stable pressure or developed persistent arrhythmias during this time were excluded from the study. After stabilization, LVP and its first derivative (dP/dt), heart rate, and coronary perfusion were measured simultaneously with a multichannel Grass 7D polygraph (Grass Instruments, Quincy, MA). Ventricular performance as a function of coronary perfusion was determined for all hearts by plotting peak systolic LVP and ±dP/dtmax values against incremental increases in coronary flow rate. Hearts were perfused with or without 20 ng/ml rMIF added to the perfusate for 20 min before readings were taken.

Determination of cardiac dysfunction by echocardiography. Echocardiograms to assess systolic function were performed using M-mode measurements. Mice were anesthetized with 5% isoflurane with 2.5 l/m O2 for 20 s (until unconscious) followed by 2% isoflurane and O2 for an average of 12–15 min. Hair was removed from the thorax and upper abdomen using Nair hair remover and gauze after sitting for 3 min. Echocardiographic measurements were obtained on anesthetized mice ~5–8 min after induction to allow any transient anesthesia-related cardiac depression to resolve. Transient, minimal changes in cardiac function detected by echocardiography have been reported after inhaled isoflurane, although fractional shortening percentage (FS%) has been reported to be stable (39). Echocardiography was performed using a Hewlett-Packard Sonos 5500 (Agilent Technologies; Edmonton, Alberta, Canada) with a frame rate of 300–500 frames/s in a random and blinded manner. A 12-MHz linear transducer was placed on the left hemithorax interfaced with a layer of US transmission gel (Aquasonic 100, Parker Laboratories; Fairfield, NJ). The two-dimensional parasternal short-axis imaging plane guided LV M-mode tracings close to the papillary muscle level. Depth was set at a minimum of 2 cm with a sweep speed of 150 m/s. Tracings were printed on a Sony color printer (UP-5200, Sony).

M-mode measurements. Data represent the average of nine selected cardiac cycles from at least two separate scans. End diastole was defined as the maximal LV diastolic dimension, and end systole was defined as the peak of posterior wall motion. FS%, a surrogate of systolic function, was calculated from LV dimensions as follows: FS% = (LVED – LVES)/LVED x 100, as shown in Fig. 5A, where LVED and LVES are LV dimensions at end diastole and end systole, respectively.



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  		Fig. 5. Echocardiographic assessment of the effects of anti-MIF antibody therapy after LPS administration demonstrates the protective effects of MIF blockade. Representative M-mode echocardiograms in wild-type mice at baseline and 8 h after LPS administration (A and B, respectively). C and D depict representative echocardiograms in LPS plus anti-MIF treated mice at 8 and 48 h, respectively. A significant recovery of cardiac function [fractional shortening percentage (FS%)] is observed in LPS-challenged mice when anti-MIF antibodies are given pretreatment (E). Data from each group represent means ± SE of 9 cardiac cycles from 3 mice monitored at multiple time points. Cardiac function determined by echocardiography is expressed as FS% [(LVED – LVES)/LVED x 100] ± SE, where LVED and LVES are LV dimensions at end diastole and end systole, respectively, and was analyzed using a 1-way repeated-measures ANOVA and multiple-comparison procedure employing the Tukey test to determine significant differences between specific groups (*P < 0.05), compared with the sham control group.

 

Determination of serum MIF levels. Sera from six mice per time point were assayed for mouse MIF using the Chemikine rat/mouse macrophage inhibitory factor (MIF) EIA kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. Briefly, 5 µl of standards, samples, or reaction buffer (blank) were added to each well in triplicate. Next, 100 µl of diluted MIF-HRP antibody conjugate were added to each well and allowed to incubate for 2 h at room temperature. Wells were then washed five times, and 100 µl of 3,3',5,5'-tetramethylbenzidene substrate were added and allowed to incubate in the dark for 30 min at room temperature. The stop reagent (0.5 N H2SO4-0.5 HCl) was added to each well and gently mixed, and the ELISA was read on an MRX Revelation microtiter plate reader (Dynex Technologies, Chantilly, VA) at 450 nm within 30 min of completion of the assay (reference at 630 nm).

Statistical analysis. Northern and Western data are expressed as means ± SE and were statistically analyzed using a one-way ANOVA. A multiple-comparison procedure was employed using the Tukey method to determine statistical significance between groups. Cardiac function determined by the Langendorff preparation (including stabilization data) is expressed as the mean ± SE, and separate analyses were performed for each parameter measured (e.g., LVP, +dP/dtmax) as a function of treatment group and coronary flow rate using a repeated-measures ANOVA. A multiple comparison procedure employing the Bonferroni method was used to determine significant differences between groups. Serum MIF levels are expressed as means ± SE and were statistically analyzed using a one-way ANOVA, with a multiple-comparison procedure employing the Bonferroni method to determine significance between groups. Cardiac function determined by M-mode echocardiography is expressed as FS% ± SE and analyzed using a one-way repeated-measures ANOVA. Additional comparisons were performed using the Tukey test to determine significant differences between specific groups. Statistical significance for all analyses was defined as P < 0.05. All statistical analyses were performed using SigmaStat 2.03 (SPSS, Chicago, IL) and Microsoft Excel (Microsoft, Seattle, WA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
MIF protein is constitutively expressed by cardiac myocytes in vivo and is released in response to LPS challenge. Both immunochemistry and Western analysis performed on cardiac tissue documented the presence of MIF in cardiac cells, including ventricular and atrial myocytes, under baseline control conditions (Figs. 1 and 2). After endotoxin challenge, both immunochemistry and immunoblot analysis document a significant decrease in cardiac tissue MIF concentration. This decrease was most profound (75% decrease) at 12 h with levels returning to near baseline control levels by 24 h. This expression pattern in the heart is similar to that witnessed in the liver and spleen (Fig. 2) and is consistent with the hypothesis that MIF is released from preformed stores within tissue after LPS challenge. The release of MIF from tissue evident at 4 h on immunoblot (Fig. 1) correlates with the increase in serum levels after endotoxin exposure (Table 1).



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  		Fig. 1. Macrophage migration inhibitory factor (MIF) is constitutively expressed in cardiac tissue and released maximally 12 h post-LPS challenge. Each data point represents the mean density in arbitrary units (AU)/mm2 ± SE of 3 independent Western blot experiments. A representative Western blot is shown below the graph. One-way ANOVA and multiple-comparison procedure using the Tukey method were employed to determine statistical significance compared with the control group (*P < 0.05). C, control.

 


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  		Fig. 2. MIF, constitutively present in the heart, liver, and spleen, is decreased after LPS challenge. Preformed MIF in the heart, liver, and spleen (A, E, I) decreases 12 h after LPS challenge (B, F, and J) and is replenished after 24 h (C, G, and K) as demonstrated by immunohistochemistry. A negative control (secondary antibody without the primary anti-MIF antibody) demonstrated that no background staining was present at each time point in each organ investigated as represented in the far right column (D, H, L). Magnification: x100 (kidney, spleen); x400 (heart).

 

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  		Table 1. Serum MIF concentration after a 4 mg/kg endotoxin challenge

 

Myocardial MIF mRNA expression increases after endotoxin challenge. Northern blot analysis of RNA obtained from the hearts of either control or LPS-challenged mice at given time points indicates that MIF mRNA is constitutively expressed in the hearts of control mice and that significant increases in MIF mRNA concentration are detectable at 48 h after LPS challenge (Fig. 3).



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  		Fig. 3. LPS challenge upregulates MIF mRNA in cardiac tissue significantly at 48 h. MIF and {beta}-actin mRNAs were detected using 32P-radiolabeled probes complementary to MIF and {beta}-actin mRNAs. Each data point represents the mean density in AU/mm2 ± SE of 3 independent Northern blot experiments. A representative Northern blot is shown below the graph. Normalized MIF was determined by the ratio of MIF density to {beta}-actin density. One-way ANOVA and multiple-comparison procedure using the Tukey method were employed to determine statistical significance compared with the control group (*P < 0.05).

 

MIF induces systolic and diastolic cardiac dysfunction in an LPS-independent mechanism. To determine if MIF directly influences cardiac function, spontaneously beating normal mouse hearts (Langendorff preparation) were perfused with rMIF at a concentration of 20 ng/ml, approximating that documented in the serum of humans with septic shock (18). The human MIF used in the Langendorff perfusion studies has been shown to have an ~90% homology with murine MIF (30) and has been shown to have cross-species biological function (5, 30). Therefore, its activity should be similar to murine MIF. Responses to MIF were determined in hearts from both C57BL/6J and C3H/HeJ mice. C3H/HeJ mice are resistant to endotoxin (35–37), thereby controlling for the possibility that any depression observed might be due to trace amount of endotoxin in the perfusate. Table 2 illustrates the responses of both mouse strains to retrograde aortic perfusion at 1.5 ml/min with control perfusate or perfusate containing 20 ng/ml rMIF after stabilization. Perfusion with MIF led to a significant decrease in LVP, +dP/dtmax, –dP/dtmax, and rate of LVP rise at a developed pressure of 40 mmHg (dP40; mmHg/s) in both mouse strains, whereas other parameters (time to maximal ±dP/dt, coronary perfusion pressure, coronary vascular resistance, and heart rate) were unaffected. Figure 4 illustrates the effect of rMIF over a range of coronary flow rates. Increases in coronary flow produced a stepwise increase in contractile performance in all hearts regardless of experimental group assignment. Comparison of the rMIF-challenged hearts with control hearts revealed a downward shift in the function curves, indicating significant systolic and diastolic depression in response to 20 ng/ml rMIF (P < 0.05). The effect of rMIF was statistically identical in both endotoxin-sensitive (C57BL/6J) and endotoxin-resistant (C3H/HeJ) strains. Likewise, there were no differences in LVP, +dP/dtmax, and –dP/dtmax between the C57BL/6J and C3H/HeJ study hearts perfused with rMIF.


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  		Table 2. In vitro stabilization data from isolated hearts in the Langendorff perfusion experiments

 


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  		Fig. 4. Cardiac function determination by Langendorff preparation post-recombinant human MIF (rMIF) perfusion in C57BL/6J mice and endotoxin-resistant C3H/HeJ mice demonstrates that rMIF mediates cardiac dysfunction in an LPS-independent mechanism. Cardiac function is expressed as means ± SE of 7 C3H/HeJ and 10 C57BL/6J independent Langendorff experiments. Separate analyses were performed for each left ventricular (LV) pressure (LVP), maximum positive and negative first derivative of LVP with respect to time (+dP/dtmax and –dP/dtmax, respectively) as a function of treatment group and coronary flow rate using a repeated-measures ANOVA and multiple-comparison procedure employing the Bonferroni method to determine significant differences between groups (*P < 0.05).

 

Anti-MIF antibodies improve LPS-induced cardiac depression in vivo. To determine the influence of MIF in the pathogenesis of cardiac dysfunction in vivo, serial echocardiography (M-mode) was performed on LPS-challenged mice that had been pretreated (90 min prior) with either of two anti-MIF monoclonal antibodies, an isotype control antibody, or no treatment (Fig. 5). At 4 h post-LPS challenge, the FS% of all LPS-challenged mice was similarly depressed (50% reduction in FS%), irrespective of group assignment. Eight hours post-LPS challenge, however, mice injected with either of the two monoclonal anti-MIF antibodies demonstrated statistically significant recovery of FS% compared with LPS-challenged groups receiving either no treatment or isotype antibody control (Fig. 5). This enhanced recovery of function persisted at 12, 24, and 48 h. At 48 h after challenge, anti-MIF-treated groups had near total restoration of FS%, whereas LPS-challenged groups receiving isotype control or no pretreatment remained profoundly depressed. Throughout the 48 h, the FS% of sham-operated mice did not change significantly, indicating that cardiac function was unaffected by anesthesia or the testing regimen itself. Additionally, at all time points, the mice injected with isotypic antibody controls were identical to animals challenged with LPS alone, indicating specificity of the anti-MIF antibody effects.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
This study is the first to demonstrate that macrophage MIF is a myocardial depressant factor and, in this regard, functions as an important late mediator of endotoxin-induced cardiac dysfunction in vivo. The timing of improved cardiac function associated with MIF blockade was consistent with the time course of MIF release from the myocardium and other tissues. Moreover, because the myocardium is itself a significant tissue source of MIF, these data raise the possibility that MIF may mediate cardiac dysfunction in other cardiac diseases.

As demonstrated in our echocardiography studies, MIF neutralization using anti-MIF antibodies confers significant protection from LPS-induced cardiac dysfunction beginning at 8 h post-LPS challenge (Fig. 5). Moreover, we have shown that rMIF induces nearly immediate cardiac depression ex vivo in the Langendorff perfusion studies (Fig. 4). Taken together, these data indicate that MIF must be locally present at the heart by 8 h after LPS challenge to mediate its cardiodepressant effects. Whether the source of this MIF is systemic or local is not entirely clear, but this study suggests that local MIF production may be important. The maximum circulating MIF levels after LPS challenge are modest, increasing only 1.5-fold by 8 h (Table 1); however, the maximum MIF release from cardiac tissue is more dramatic, decreasing fourfold by 12 h (Fig. 1). Because the timing and magnitude of MIF release from the heart itself is more impressive then systemic changes in MIF, local MIF release from the heart is more likely to be responsible for the cardiodepressant effects.

Further evidence that myocardial MIF works in an autocrine fashion to induce cardiac dysfunction after LPS challenge is demonstrated in the timing of maximal protection from LPS-induced cardiac dysfunction provided by MIF neutralization using anti-MIF antibodies (Fig. 5). Relatively minimal, although significant, recovery of heart function occurs at 8 h post-LPS challenge when circulating serum MIF levels are highest. However, maximal recovery of cardiac function is observed at 48 h post-LPS challenge when serum levels have returned to baseline. The time course for the recovery of heart function more closely correlates with the maximal release of MIF from the myocardium, although MIF's immediate effect in vitro is not likely the only effect expected as prolonged effects in vivo have been reported (28, 32). Similar local (autocrine) effects have been described with myocardial TNF-{alpha} production. Specifically, transgenic mice that overproduce TNF-{alpha} specifically in the heart develop a reproducible cardiomyopathy (20).

Previous studies have demonstrated a greater than five- to sixfold increase in serum MIF by ~2 h post-LPS challenge (9). In this study, both the maximum increase in serum MIF (1.5-fold of baseline vs. 5- to 6-fold previously reported) and the timing of MIF release into the serum (maximum at 8 h vs. 2 h previously reported) differ from previous reports. Possible reasons for this relatively blunted and slightly delayed response include strain differences among mice, the LPS dosage, and differences in assay sensitivities. Strain differences in MIF responses have been previously reported, specifically between C57BL/6J and BALB/c mice (1). Cells from BALB/c mice were shown to consistently secrete significantly higher levels of MIF in response to various stimuli compared with other stains (including C57BL/6J) (1). The MIF serum response may also differ because of the higher dose (4 mg/kg) used in this study compared with the previous report in BALB/c mice (9). Prior studies have demonstrated that increasing concentrations of LPS blunt MIF production by macrophages (9). The reason for the relatively high serum "background" MIF concentration determined by ELISA compared with previous determinations by Western blot is not clear but has been reported by other investigators using the same ELISA kit (45). Great care was taken to avoid obvious causes of falsely increased MIF levels (as reported by the manufacturer) by ELISA such as hemolysis. Indirect evidence supporting the proposal that our baseline MIF levels are falsely elevated comes from echocardiographic studies. When healthy wild-type (C57BL/6J) mice are given anti-MIF antibodies alone, no increase in function is noted by echocardiography (unpublished data). This suggests that baseline MIF levels are not very high and/or that circulating MIF alone may not be sufficient to cause myocardial depressant effects. Indeed, other cofactors such TNF-{alpha} or LPS may be necessary in order for MIF to mediate cardiac dysfunction in vivo.

Cardiac dysfunction during sepsis (12, 22) is associated with poor outcome in both humans (2, 40) and animals (7, 11). We and others (19, 21, 23) have previously demonstrated that sepsis- or burn-associated cardiac dysfunction is primarily due to circulating myocardial depressant factors, including TNF-{alpha}. However, because TNF-{alpha} is a sentinel, rapid-response cytokine and is gone from the circulation days or weeks before the resolution of myocardial dysfunction, we entertained the possibility that additional important myocardial depressant proteins might exist. When preliminary microarray data on cardiac gene expression highlighted that MIF is expressed in cardiac tissue (not published), we entertained the hypothesis that MIF itself might be a myocardial depressant protein. It has already been well established that MIF plasma levels are significantly elevated in patients with sepsis or systemic inflammatory response syndrome (18, 25) and that the kinetics of MIF release occur several hours after the initial cardiac dysfunction after LPS challenge is observed. These data supported the hypothesis that MIF could be a myocardial depressant protein that might account for late, prolonged cardiac depression during sepsis. Indeed, our results indicate that MIF perfusion directly depresses cardiac function in vitro; moreover, treatment with either of two independent monoclonal antibodies directed against MIF mitigates late myocardial depression in our model.

Studies utilizing live bacteria, either by direct intraperitoneal injection of E. coli or by cecal ligation and puncture (CLP), have previously demonstrated that MIF plasma and/or peritoneal fluid levels increase several hours postchallenge and that antibodies against MIF protected these mice from lethal bacterial peritonitis (11). Interestingly, mice were protected when the antibodies were given as late as 8 h after the onset of infection (11). In the present study, evidence supporting a delayed release was seen by Western blot and immunohistochemistry, which both demonstrated significant release of MIF from cardiac, liver, and spleen tissue 12 h after LPS challenge. Indirect support for this delayed MIF release is also evidenced by the delayed onset of cardiac protection beginning at 8 h post-LPS challenge and continuing thereafter. The delayed release of MIF after LPS challenge makes MIF an interesting potential therapeutic target.

MIF has a number of properties that make it unique among cytokines. MIF is released preformed from numerous cell types, including the lymphocytes, macrophages, and the anterior pituitary (5, 10, 15, 27). However, the list of sources of MIF continues to grow and includes other tissues such as the heart, lung, liver, adrenal, spleen, kidney, skin, muscle, thymus, skin, and testes (4, 16, 45). MIF has at least two catalytic activities that are distinct: tautomerase and oxidoreductase activity. To this end, pharmacological inhibitors of MIF tautomerase activity have been developed for the treatment of MIF-related diseases such as sepsis, acute respiratory distress syndrome, asthma, atopic dermatitis, rheumatoid arthritis, nephropathy, and cancer (14, 33). Most of these diseases have shown benefit from treatment with anti-MIF antibodies, at least in animal models; whether specific inhibition of tautomerase or oxidoreductase enzymatic activity might yield similar benefits remains untested.

We have not yet determined the precise mechanism by which MIF affects cardiac function. However, several investigations indicate that MIF may exert effects by both direct and indirect mechanisms. Previous studies have provided evidence that MIF promotes the release and pharmacodynamic effects of other proinflammatory cytokines. Macrophages expressing anti-sense MIF cDNA (leading to less endogenous MIF) secrete/express significantly less TNF-{alpha}, IL-6, and nitric oxide (NO), while NF-{kappa}B activity is decreased in response to LPS (38). Therefore, it appears that MIF may directly interact with the LPS signaling pathway by unknown mechanisms (26). Moreover, MIF knockout mice, which are resistant to lethal doses of LPS, have lower circulating plasma levels of TNF-{alpha} compared with wild-type mice at baseline. On LPS challenge, these mice demonstrate diminished circulating TNF-{alpha} concentrations, increased NO concentrations, and unchanged IL-6 and IL-12 concentrations (7). While MIF appears to promote proinflammatory cytokines, the effects of MIF have been shown to act in a TNF-{alpha}-independent manner in sepsis. When CLP was performed in TNF-{alpha} knockout mice, a 60% survival rate (at 15 h) was seen in mice administered anti-MIF antibodies compared with a 0% survival rate in TNF-{alpha} knockout mice (11).

In relation to cardiac dysfunction unrelated to sepsis, elevated serum MIF concentrations have been described in patients after acute myocardial infarction (42, 43, 46) with heretofore unknown physiological relevance. In addition, increased tissue expression of MIF occurs in the myocardium following a model of acute myocardial infarction in the rat (45). Similarly, cultured cardiac myocytes have been noted to release MIF in response to hypoxia and hydrogen peroxide (free radical initiator) but not angiotensin II, endothelin-1, IL-1{beta}, or TNF-{alpha} (17, 43). Taken together, these results suggest that there are many clinical scenarios that could potentially trigger myocardial MIF release, thereby adversely affecting cardiac function, for which anti-MIF therapies may be of benefit.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Institutes of Health Grant RO1-GM-58863.


   ACKNOWLEDGMENTS
 
We acknowledge the Univ. of Texas Southwestern Dept. of Pathology Immunohistochemistry Laboratory for assistance with immunohistochemistry and Cytokine PharmaSciences for providing the monoclonal antibodies utilized in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. P. Giroir, Dept. of Pediatrics, Univ. of Texas Southwestern, 5323 Harry Hines Blvd., Dallas, TX 75390-9063 (E-mail: brett.giroir{at}childrens.com).

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.

* L. B. Garner and M. S. Willis contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Adelman N, Cohen S, and Yoshida T. Strain variations in murine MIF production. J Immunol 121: 209–212, 1978.[Abstract/Free Full Text]
  2. Ammann P, Fehr T, Minder EI, Gunter C, and Bertel O. Elevation of troponin I in sepsis and septic shock. Intensive Care Med 27: 965–969, 2001.[ISI][Medline]
  3. Apte RS, Sinha D, Mayhew E, Wistow GJ, and Niederkorn JY. Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege. J Immunol 160: 5693–5696, 1998.[Abstract/Free Full Text]
  4. Bacher M, Meinhardt A, Lan HY, Mu W, Metz CN, Chesney JA, Calandra T, Gemsa D, Donnelly T, Atkins RC, and Bucala R. Migration inhibitory factor expression in experimentally induced endotoxemia. Am J Pathol 150: 235–246, 1997.[Abstract]
  5. Bernhagen J, Calandra T, and Bucala R. Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features. J Mol Med 76: 151–161, 1998.[ISI][Medline]
  6. Bernhagen J, Calandra T, Mitchell RA, Martin SB, Tracey KJ, Voelter W, Manogue KR, Cerami A, and Bucala R. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature 365: 756–759, 1993.[Medline]
  7. Bozza M, Satoskar AR, Lin G, Lu B, Humbles AA, Gerard C, and David JR. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J Exp Med 189: 341–346, 1999.[Abstract/Free Full Text]
  8. Bryant D, Becker L, Richardson J, Shelton J, France F, Peshock R, Thompson M, and Giroir BP. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-{alpha} (TNF). Circulation 97: 1375–1381, 1998.[Abstract/Free Full Text]
  9. Calandra T, Bernhagen J, Mitchell RA, and Bucala R. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J Exp Med 179: 1895–1902, 1994.[Abstract/Free Full Text]
  10. Calandra T and Bucala R. Macrophage migration inhibitory factor (MIF): a glucocorticoid counter-regulator within the immune system. Crit Rev Immunol 17: 77–88, 1997.[ISI][Medline]
  11. Calandra T, Echtenacher B, Roy DL, Pugin J, Metz CN, Hultner L, Heumann D, Mannel D, Bucala R, and Glauser MP. Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nat Med 6: 164–170, 2000.[ISI][Medline]
  12. Court O, Kumar A, and Parrillo JE. Clinical review: myocardial depression in sepsis and septic shock. Crit Care 6: 500–508, 2002.[ISI][Medline]
  13. De Jong YP, Abadia-Molina AC, Satoskar AR, Clarke K, Rietdijk ST, Faubion WA, Mizoguchi E, Metz CN, Alsahli M, ten Hove T, Keates AC, Lubetsky JB, Farrell RJ, Michetti P, van Deventer SJ, Lolis E, David JR, Bhan AK, Terhorst C, and Sahli MA. Development of chronic colitis is dependent on the cytokine MIF. Nat Immun 2: 1061–1066, 2001.[ISI][Medline]
  14. Dios A, Mitchell RA, Aljabari B, Lubetsky J, O'Connor K, Liao H, Senter PD, Manogue KR, Lolis E, Metz C, Bucala R, Callaway DJ, and Al-Abed Y. Inhibition of MIF bioactivity by rational design of pharmacological inhibitors of MIF tautomerase activity. J Med Chem 45: 2410–2416, 2002.[ISI][Medline]
  15. Donnelly SC and Bucala R. Macrophage migration inhibitory factor: a regulator of glucocorticoid activity with a critical role in inflammatory disease. Mol Med Today 3: 502–507, 1997.[ISI][Medline]
  16. Fingerle-Rowson G, Koch P, Bikoff R, Lin X, Metz CN, Dhabhar FS, Meinhardt A, and Bucala R. Regulation of macrophage migration inhibitory factor expression by glucocorticoids in vivo. Am J Pathol 162: 47–56, 2003.[Abstract/Free Full Text]
  17. Fukuzawa J, Nishihira J, Hasebe N, Haneda T, Osaki J, Saito T, Nomura T, Fujino T, Wakamiya N, and Kikuchi K. Contribution of macrophage migration inhibitory factor to extracellular signal-regulated kinase activation by oxidative stress in cardiomyocytes. J Biol Chem 277: 24889–24895, 2002.[Abstract/Free Full Text]
  18. Gando S, Nishihira J, Kobayashi S, Morimoto Y, Nanzaki S, and Kemmotsu O. Macrophage migration inhibitory factor is a critical mediator of systemic inflammatory response syndrome. Intensive Care Med 27: 1187–1193, 2001.[ISI][Medline]
  19. Giroir BP, Horton JW, White DJ, McIntyre KL, and Lin CQ. Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock. Am J Physiol Heart Circ Physiol 267: H118–H124, 1994.[Abstract/Free Full Text]
  20. Graciano AL, Bryant DD, White DJ, Horton J, Bowles NE, and Giroir BP. Targeted disruption of ICAM-1, P-selectin genes improves cardiac function and survival in TNF-{alpha} transgenic mice. Am J Physiol Heart Circ Physiol 280: H1464–H1471, 2001.[Abstract/Free Full Text]
  21. Haudek SB, Bryant DD, and Giroir BP. Differential regulation of myocardial NF{kappa}B following acute or chronic TNF-{alpha} exposure. J Mol Cell Cardiol 33: 1263–1271, 2001.[ISI][Medline]
  22. Krishnagopalan S, Kumar A, and Parrillo JE. Myocardial dysfunction in the patient with sepsis. Curr Opin Crit Care 8: 376–388, 2002.[Medline]
  23. Kumar A, Thota V, Dee L, Olson J, Uretz E, and Parrillo JE. Tumor necrosis factor {alpha} and interleukin 1{beta} are responsible for in vitro myocardial cell depression induced by human septic shock serum. J Exp Med 183: 949–958, 1996.[Abstract/Free Full Text]
  24. Leech M, Metz C, Santos L, Peng T, Holdsworth SR, Bucala R, and Morand EF. Involvement of macrophage migration inhibitory factor in the evolution of rat adjuvant arthritis. Arthritis Rheum 41: 910–917, 1998.[ISI][Medline]
  25. Lehmann LE, Novender U, Schroeder S, Pietsch T, von Spiegel T, Putensen C, Hoeft A, and Stuber F. Plasma levels of macrophage migration inhibitory factor are elevated in patients with severe sepsis. Intensive Care Med 27: 1412–1415, 2001.[ISI][Medline]
  26. Lue H, Kleemann R, Calandra T, Roger T, and Bernhagen J. Macrophage migration inhibitory factor (MIF): mechanisms of action and role in disease. Microbes Infect 4: 449–460, 2002.[ISI][Medline]
  27. Mitchell RA and Bucala R. Tumor growth-promoting properties of macrophage migration inhibitory factor (MIF). Semin Cancer Biol 10: 359–366, 2000.[ISI][Medline]
  28. Mitchell RA, Liao H, Chesney J, Fingerle-Rowson G, Baugh J, David J, and Bucala R. Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: regulatory role in the innate immune response. Proc Natl Acad Sci USA 99: 345–350, 2002.[Abstract/Free Full Text]
  29. Mitchell RA, Metz CN, Peng T, and Bucala R. Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). Regulatory role in cell proliferation and glucocorticoid action. J Biol Chem 274: 18100–18106, 1999.[Abstract/Free Full Text]
  30. Mozetic-Francky B, Cotic V, Ritonja A, Zerovnik E, and Francky A. High-yield expression and purification of recombinant human macrophage migration inhibitory factor. Protein Expr Purif 9: 115–124, 1997.[ISI][Medline]
  31. Natanson C, Eichenholz PW, Danner RL, Eichacker PQ, Hoffman WD, Kuo GC, Banks SM, MacVittie TJ, and Parillo JE. Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med 169: 823–932, 1989.[Abstract/Free Full Text]
  32. Onodera S, Nishihira J, Iwabuchi K, Koyama Y, Yoshida K, Tanaka S, and Minami A. Macrophage migration inhibitory factor up-regulates matrix metalloproteinase-9 and -13 in rat osteoblasts. Relevance to intracellular signaling pathways. J Biol Chem 277: 7865–7874, 2002.[Abstract/Free Full Text]
  33. Orita M, Yamamoto S, Katayama N, and Fujita S. Macrophage migration inhibitory factor and the discovery of tautomerase inhibitors. Curr Pharm Des 8: 1297–1317, 2002.[ISI][Medline]
  34. Parker MM, McCarthy KE, Ognibene FP, and Parillo JE. Right ventricular dysfunction and dilatation, similar to left ventricular changes, characterize the cardiac depression of septic shock in humans. Chest 97: 126–131, 1990.[Abstract/Free Full Text]
  35. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, and Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085–2088, 1998.[Abstract/Free Full Text]
  36. Poltorak A, Smirnova I, He X, Liu MY, Van Huffel C, McNally O, Birdwell D, Alejos E, Silva M, Du X, Thompson P, Chan EK, Ledesma J, Roe B, Clifton S, Vogel SN, and Beutler B. Genetic and physical mapping of the Lps locus: identification of the toll-4 receptor as a candidate gene in the critical region. Blood Cells Mol Dis 24: 340–355, 1998.[ISI][Medline]
  37. Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, and Malo D. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med 189: 615–625, 1999.[Abstract/Free Full Text]
  38. Roger T, David J, Glauser MP, and Calandra T. MIF regulates innate immune responses through modulation of Toll-like receptor 4. Nature 414: 920–924, 2001.[Medline]
  39. Roth DM, Swaney JS, Dalton ND, Gilpin EA, and Ross J Jr. Impact of anesthesia on cardiac function during echocardiography in mice. Am J Physiol Heart Circ Physiol 282: H2134–H2140, 2002.[Abstract/Free Full Text]
  40. Sessler CN and Shepherd W. New concepts in sepsis. Curr Opin Crit Care 8: 465–472, 2002.[Medline]
  41. Suffredini AF, Fromm RE, Parker MM, Brenner M, Kovacs JA, Wesley RA, and Parillo JE. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med 321: 280–287, 1989.[Abstract]
  42. Takahashi M, Nishihira J, Katsuki T, Kobayashi E, Ikeda U, and Shimada K. Elevation of plasma levels of macrophage migration inhibitory factor in patients with acute myocardial infarction. Am J Cardiol 89: 248–249, 2002.[ISI][Medline]
  43. Takahashi M, Nishihira J, Shimpo M, Mizue Y, Ueno S, Mano H, Kobayashi E, Ikeda U, and Shimada K. Macrophage migration inhibitory factor as a redox-sensitive cytokine in cardiac myocytes. Cardiovasc Res 52: 438–445, 2001.[Abstract/Free Full Text]
  44. White J, Maass DL, Giroir B, and Horton JW. Development of an acute burn model in adult mice for studies of cardiac function and cardiomyocyte cellular function. Shock 16: 122–129, 2001.[ISI][Medline]
  45. Yu CM, Lai KW, Chen YX, Huang XR, and Lan HY. Expression of macrophage migration inhibitory factor in acute ischemic myocardial injury. J Histochem Cytochem 51: 625–631, 2003.[Abstract/Free Full Text]
  46. Yu CM, Lau CP, Lai KW, Huang XR, Chen WH, and Lan HY. Elevation of plasma level of macrophage migration inhibitory factor in patients with acute myocardial infarction. Am J Cardiol 88: 774–777, 2001.[ISI][Medline]



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