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*L-TYROSINE
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*Sepsis
The Journal of Immunology, 2003, 171: 6827-6837.
Copyright © 2003 by The American Association of Immunologists

Peroxisome Proliferator Activator Receptor-{gamma} Ligands, 15-Deoxy-{Delta}12,14-Prostaglandin J2 and Ciglitazone, Reduce Systemic Inflammation in Polymicrobial Sepsis by Modulation of Signal Transduction Pathways 1

Basilia Zingarelli2,*, Maeve Sheehan*, Paul W. Hake*, Michael O’Connor*, Alvin Denenberg* and James A. Cook{dagger}

* Division of Critical Care, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and {dagger} Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, SC 29425


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Peroxisome proliferator activator receptor-{gamma} (PPAR{gamma}) is a nuclear receptor that controls the expression of several genes involved in metabolic homeostasis. We investigated the role of PPAR{gamma} during the inflammatory response in sepsis by the use of the PPAR{gamma} ligands, 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) and ciglitazone. Polymicrobial sepsis was induced by cecal ligation and puncture in rats and was associated with hypotension, multiple organ failure, and 50% mortality. PPAR{gamma} expression was markedly reduced in lung and thoracic aorta after sepsis. Immunohistochemistry showed positive staining for nitrotyrosine and poly(ADP-ribose) synthetase in thoracic aortas. Plasma levels of TNF-{alpha}, IL-6, and IL-10 were increased. Elevated activity of myeloperoxidase was found in lung, colon, and liver, indicating a massive infiltration of neutrophils. These events were preceded by degradation of inhibitor {kappa}B{alpha} (I{kappa}B{alpha}), activation of I{kappa}B kinase complex, and c-Jun NH2-terminal kinase and, subsequently, activation of NF-{kappa}B and AP-1 in the lung. In vivo treatment with ciglitazone or 15d-PGJ2 ameliorated hypotension and survival, blunted cytokine production, and reduced neutrophil infiltration in lung, colon, and liver. These beneficial effects of the PPAR{gamma} ligands were associated with the reduction of I{kappa}B kinase complex and c-Jun NH2-terminal kinase activation and the reduction of NF-{kappa}B and AP-1 DNA binding in the lung. Furthermore, treatment with ciglitazone or 15d-PGJ2 up-regulated the expression of PPAR{gamma} in lung and thoracic aorta and abolished nitrotyrosine formation and poly(ADP-ribose) expression in aorta. Our data suggest that PPAR{gamma} ligands attenuate the inflammatory response in sepsis through regulation of the NF-{kappa}B and AP-1 pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Sepsis is a systemic response to infection characterized by hemodynamic and metabolic derangement that may result in septic shock, multiple organ system failure, and death. Although antibiotic therapy may effectively treat an underlying infection, this treatment is not sufficient to reverse the systemic inflammation and its consequences (1). Current evidence suggests that components of the bacteria initiate a cascade of events by stimulating monocytes, neutrophils, macrophages, or endothelial cells of the host, leading to the production and release of numerous endogenous proinflammatory mediators (2). This production is regulated at the transcriptional level by a rapid activation of the enhancer elements NF-{kappa}B (3) and AP-1, through interactions with the protein kinases I{kappa}B kinase complex (IKK), 3 and c-Jun NH2-terminal kinase (JNK), respectively (3, 4, 5).

Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) is a member of the nuclear subfamily of transcription factors with pleiotropic effects on lipid and glucose homeostasis, cell proliferation, and control of inflammation (6). The class of insulin-sensitizing drugs known as thiazolidinediones has been identified as specific PPAR{gamma} agonists, which has allowed characterization of many genes regulated by PPAR{gamma} (7, 8). Thiazolidinediones include rosiglitazone, pioglitazone, troglitazone, and ciglitazone (9). In addition to these synthetic agonists, PGs of the J2 series have been identified as natural ligands for PPAR{gamma} (10, 11). Several in vitro studies have demonstrated that pharmacological activation of PPAR{gamma} by 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) or thiazolidinediones has anti-inflammatory effects. For example, the PPAR{gamma} ligands 15d-PGJ2 and rosiglitazone repressed the expression of several inflammatory response genes in activated macrophages, including the genes encoding inducible NO synthase (iNOS), TNF-{alpha}, gelatinase B, and cyclooxygenase 2 (COX-2) (12, 13). Similarly, 15d-PGJ2 and troglitazone inhibited the production of TNF-{alpha}, IL-6, and IL-1{beta} in activated human monocytes (13). In line with these findings, in vivo treatment with PPAR{gamma} agonists has been reported to attenuate the inflammatory process of experimental colitis in mice (14, 15) and adjuvant-induced arthritis in rats (16).

Considering these anti-inflammatory properties of PPAR{gamma}, we investigated the biological effects of two chemically unrelated ligands, 15d-PGJ2 and ciglitazone, in the inflammatory response of sepsis. Specifically, we determined the extent of hemodynamic derangement and tissue damage in a rat model of polymicrobial sepsis. Furthermore, to gain a better understanding of the regulatory role of PPAR{gamma}, we investigated whether in vivo treatment with 15d-PGJ2 and ciglitazone may affect the proinflammatory signal transduction mechanisms of sepsis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Rat model of cecal ligation and puncture (CLP)

The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and commenced with the approval of the institutional animal care and use committee. Male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 175–250 g, were anesthetized with thiopentone sodium (70 mg/kg) i.p. CLP was performed as previously described (17). After opening the abdomen, the cecum was exteriorized and ligated by a 3.0 silk ligature at its base without obstructing intestinal continuity. The cecum was punctured twice with an 18-gauge needle and returned to the peritoneal cavity. The abdominal incision was closed with 3-silk running suture.

Six groups of rats were used in the experiment. The first group (n = 12) received an equal volume of vehicle (0.9% NaCl solution) instead of the PPAR{gamma} ligands (CLP + vehicle group). The second and third groups (n = 12 for each group) received 15d-PGJ2 (1 mg/kg; CLP + 15d-PGJ2 group) or ciglitazone (5 mg/kg; CLP + ciglitazone group) by i.p. injection. In the fourth group of rats surgery was performed as in the CLP group, but the cecum was neither ligated nor punctured (sham + vehicle group; n = 10). In another set of experiments, the effect of treatment with 15d-PGJ2 and ciglitazone was examined in control rats (i.e., not subjected to CLP; n = 4; sham + 15d-PGJ2 and sham + ciglitazone groups) to rule out nonspecific effects of PPAR{gamma} ligands. Drugs were administered at 1, 6, and 12 h after CLP and every 12 h thereafter until the end of the observation period. Saline solution (0.9%, 5 ml) was given s.c. to replace the fluid and blood loss during the operation. Groups of animals (n = 3–12) were sacrificed at different time points after CLP (1, 3, 6, and 18 h). Plasma samples, lungs, colons, livers, and aortas were collected for the immunohistochemical and biochemical studies described below. In other groups of rats (n = 17–22 for each group) survival was monitored for 4 days.

Hemodynamic measurements

Mean arterial blood pressure was measured in other groups of rats (n = 4–10 for each group). In the first experiment rats were anesthetized with thiopentone sodium (70 mg/kg i.p.). The trachea was cannulated to facilitate respiration, and the carotid artery was cannulated to measure mean arterial blood pressure by a pressure transducer connected to a Maclab A/D converter (AD Instruments, Milford, MA). In these anesthetized rats mean arterial blood pressure was then monitored for 6 h after induction of CLP. In the second experiment animals underwent CLP and were allowed to recover. Fourteen hours later, the animals were anesthetized, and the trachea and carotid artery were cannulated. In these anesthetized rats mean arterial blood pressure was measured for 4 h (i.e., up to 18 h after CLP). Animals that died before the end of the experiment were excluded from the study.

White blood cell count, measurement of bacterial clearance, and glucose levels

Standard techniques of clinical pathology were used to determine white blood cell count, bacterial presence in hemocultures, and blood glucose levels.

Immunohistochemistry for PPAR{gamma}, nitrotyrosine, and poly(ADP-ribose) synthetase (PARS)

Immunohistochemistry was used to evaluate the expression of PPAR{gamma}, tyrosine nitration, a marker of nitrosative damage, and the activation of PARS, a nuclear enzyme involved in the inflammatory process of sepsis. Paraffin-embedded sections (5 µm) of lung and thoracic aorta were deparaffinized, treated with 0.3% hydrogen peroxide for 15 min to block endogenous peroxidase activity, and then rinsed briefly in PBS. Nonspecific binding was blocked by incubating the slides with a blocking solution (0.1 M PBS containing 0.1% Triton X-100 and 2% normal goat serum) for 2 h. Sections were incubated overnight with primary anti-PPAR{gamma}1, anti-nitrotyrosine, or anti-PARS Ab or with control solutions. Control sections included buffer alone or nonspecific purified rabbit IgG. Immunoreactivity was detected with a biotinylated goat anti-rabbit secondary Ab and the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Color was developed using diaminobenzidine (18). Immunohistochemical staining was estimated by an independent pathologist blinded to the experimental protocol.

Measurement of myeloperoxidase activity

Myeloperoxidase activity was determined as an index of neutrophil accumulation in lung, colon, and liver, as previously described (18). Tissues were homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and were centrifuged for 30 min at 20,000 x g at 4°C. An aliquot of the supernatant was allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured by spectrophotometry at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol hydrogen peroxide/min at 37°C and was expressed in units per 100 mg of tissue.

Measurement of plasma levels of cytokines

Plasma levels of TNF-{alpha}, IL-6, and IL-10 were evaluated by commercially available, solid phase, sandwich ELISA kits (R&D Systems, Minneapolis, MN) using the protocol recommended by the manufacturer.

Subcellular fractionation and nuclear protein extraction

Lung samples were homogenized with a Polytron homogenizer (Brinkmann Instruments, West Orange, NY) in a buffer containing 0.32 M sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM 2-ME, 20 µM leupeptin, 0.15 µM pepstatin A, 0.2 mM PMSF, 50 mM NaF, 1 mM sodium orthovanadate, and 0.4 nM microcystin. The homogenates were centrifuged (1,000 x g, 10 min), and the supernatant (cytosol plus membrane extract) was collected for evaluation of inhibitor {kappa}B{alpha} (I{kappa}B{alpha}) content and IKK activity as described below. The pellets were solubilized in Triton buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.2 mM sodium orthovanadate, 20 µM leupeptin A, and 0.2 mM PMSF). The lysates were centrifuged (15,000 x g, 30 min, 4°C), and the supernatant (nuclear extract) was collected for evaluation of PPAR{gamma} content, JNK activity, and DNA binding of NF-{kappa}B and AP-1.

Western blot analysis for PPAR{gamma} and I{kappa}B{alpha}

The nuclear content of PPAR{gamma} and cytosol degradation of I{kappa}B{alpha} in the lung were determined by immunoblot analyses. Nuclear and cytosol extracts were boiled in equal volumes of loading buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 10% 2-ME), and 50 µg of protein was loaded per lane on an 8–16% Tris-glycine gradient gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked with 5% nonfat dried milk in TBS for 1 h and then were incubated with primary Abs against PPAR{gamma} or I{kappa}B{alpha} for 1 h. The membranes were washed in TBS with 0.1% Tween 20 and incubated with secondary peroxidase-conjugated Ab. Immunoreaction was visualized by chemiluminescence. Densitometric analysis of blots was performed using ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Assay of IKK and JNK activities

IKK and JNK activities were determined by immune complex kinase assay and were estimated as the ability to phosphorylate GST-I{kappa}B{alpha} or GST-c-Jun, respectively (18). After immunoprecipitation of lysate with specific Ab directed to IKK{gamma} or JNK1, the immunoprecipitate was incubated for 30 min at 30°C in 40 µl of reaction buffer (25 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM glycerol phosphate, 0.1 mM sodium orthovanadate, 2 mM DTT, 25 µM ATP, and 5 µCi [{gamma}-32P]ATP). GST-I{kappa}B{alpha}1–54 (1 µg) was used as substrate for the IKK complex; GST-c-Jun1–79 (1 µg) was used as substrate for JNK. Reaction products were separated by SDS-PAGE and visualized by autoradiography. Densitometric analysis was performed using ImageQuant (Molecular Dynamics).

EMSA

EMSAs were performed as previously described (18). Oligonucleotide probes corresponding to the NF-{kappa}B consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') or the AP-1 consensus sequence (5'-CGC TTG ATG ACT CAG CCG GAA-3') were labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase and were purified in Bio-Spin chromatography columns (Bio-Rad, Hercules, CA). Ten micrograms of nuclear protein were preincubated with EMSA buffer (12 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH 7.9), 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 50 ng/ml poly(d(I-C)), 12% glycerol (v/v), and 0.2 mM PMSF) on ice for 10 min before addition of the radiolabeled oligonucleotide 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 were run in 0.5x TBE (45 mM Tris-HCl, 45 mM boric acid, and 1 mM EDTA) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3M paper (Clifton, NJ), dried under a vacuum at 80°C for 1 h, and exposed to photographic film at -70°C with an intensifying screen. Densitometric analysis was performed using ImageQuant (Molecular Dynamics).

Materials

Primary anti-nitrotyrosine Ab was purchased from Upstate Biotechnology (Saranac Lake, NY). The primary Abs directed at I{kappa}B{alpha}, JNK1, and IKK{gamma} and the oligonucleotides for NF-{kappa}B and AP-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Ciglitazone, 15d-PGJ2, and the primary Abs directed at PARS and PPAR{gamma}1 were obtained from Biomol (Plymouth Meeting, PA). Reagents and secondary and nonspecific IgG Abs for immunohistochemical analysis were obtained from Vector Laboratories. The ELISA kits for TNF-{alpha}, IL-6, and IL-10 were obtained from R&D Systems. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Data analysis

All values in the figures and text are expressed as the mean ± SEM of n observations (n = 3–12 animals for each group). The results were examined by ANOVA, followed by Bonferroni’s correction post hoc t test. Fisher’s exact test was used to compare differences in survival rates. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Effect of PPAR{gamma} ligands on survival, white blood cell count, bacteremia, and glycemia in septic rats

To imitate the clinical scenario of sepsis, rats were subjected to CLP, a procedure that causes peritonitis. As expected after CLP, vehicle-treated rats exhibited signs of septicemia, and 50% of the animals died within 84–96 h (Fig. 1). At 18 h after CLP, blood cultures indicated the presence of Gram-positive bacteria such as Enterococcus, Morganella morganii, and Staphylococcus coagulase negative; Gram-negative bacteria such as Escherichia coli and Pseudomonas pickettii; and fungus such as Fusarium. The total microbial count was 2.6 x 103 colonies/ml in vehicle-treated rats. This polymicrobial septicemia was associated with a marked increase in neutrophils and reduction of lymphocytes (Table I). In contrast, rats treated with 15d-PGJ2 or ciglitazone appeared healthier. Treatment with 15d-PGJ2 or ciglitazone increased the survival rate, and 82 and 65% of animals, respectively, were still alive at 96 h (Fig. 1). The difference in survival rate was statistically significant between vehicle- and 15d-PGJ2-treated rats, but not between vehicle- and ciglitazone-treated rats. PPAR{gamma} ligands significantly reduced the level of circulating neutrophils and increased the level of lymphocytes (Table I), but did not modify blood counts of microbial colonies, which showed similar contents of Gram-negative and Gram-positive bacteria and fungus as the vehicle-treated group. Microbial counts were 2.6 x 103 and 1.6 x 103 colonies/ml in 15d-PGJ2- or ciglitazone-treated rats, respectively.



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  		FIGURE 1. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on survival rate in rats subjected to CLP. *, p < 0.05 vs vehicle-treated rats. 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) was administered 1, 6, and 12 h after CLP and every 12 h thereafter.

 

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  		Table I. Effects of 15d-PGJ2 and ciglitazone on blood counts of total white cells, neutrophils, and lymphocytes at 18 h after CLPa

 
Because a major physiological function of PPAR{gamma} is the regulation of glucose metabolism, we sought to ascertain whether in vivo treatment with 15d-PGJ2 or ciglitazone could affect blood glucose levels. At 18 h after CLP, only one rat of six in the vehicle-treated group appeared hyperglycemic (glucose, 227.8 mg/dl), whereas glucose levels remained similar to control values in five of six rats in the vehicle-treated group (120.7 ± 5.9 mg/dl). Glucose levels were also normal in 15d-PGJ2-treated rats (135.9 ± 2.9 mg/dl) and ciglitazone-treated rats (125.2 ± 4.8 mg/dl). The glucose level in sham control animals was 150.2 ± 13.5 mg/dl.

PPAR{gamma} ligands improve mean arterial blood pressure

Because a serious consequence of septic shock is the occurrence of hemodynamic changes, we also determined the effect of 15d-PGJ2 and ciglitazone on mean arterial blood pressure. In vehicle-treated rats, CLP resulted in a significant decrease in blood pressure starting 4 h after CLP compared with the basal level at time zero. Treatment with 15d-PGJ2 or ciglitazone significantly improved blood pressure (Fig. 2A). Treatment with 15d-PGJ2 or ciglitazone did not modify mean arterial blood pressure in control rats (i.e., not subjected to CLP; Fig. 2B).



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  		FIGURE 2. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on mean arterial blood pressure (MABP) in rats subjected to CLP (A) or in control rats (B). Each data point represents the mean ± SEM of four to 10 animals for each group. =, p < 0.05 vs time zero in the vehicle-treated group subjected to CLP; #, p < 0.05 vs vehicle-treated rats. 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) was administered 1, 6, and 12 h after CLP.

 
Polymicrobial sepsis is associated with down-regulation of PPAR{gamma} expression in lung and thoracic aortas

We next investigated whether induction of polymicrobial sepsis was associated with changes in PPAR{gamma} expression. At immunohistochemical evaluation, the expression of PPAR{gamma} was mainly localized in the epithelium of bronchi (Fig. 3B) and in the endothelium and smooth muscle cells of thoracic aortas (Fig. 4A) in control sham rats. By Western blot analysis, we observed that the nuclear content of PPAR{gamma} decreased in a time-dependent manner in lungs of vehicle-treated rats subjected to CLP (Fig. 3A). At immunohistochemical evaluation, the expression of PPAR{gamma} appeared reduced in bronchial epithelium (Fig. 3C) and in aortic endothelial and smooth muscle cells (Fig. 4B) of vehicle-treated rats 18 h after CLP. This reduction of PPAR{gamma} expression was reversed by in vivo treatment with 15d-PGJ2 and, to a partial extent, with ciglitazone (Figs. 3 and 4).



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  		FIGURE 3. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on PPAR{gamma} expression in the lung. A, Western blot analysis (top panel) of PPAR{gamma} in nuclear extracts of vehicle-treated, ciglitazone-treated, and 15d-PGJ2-treated rats 0, 1, 3, 6, and 18 h after CLP and relative densitometric analysis (lower panel) from the immunoblot. Data are representative of three separate experiments. *, p < 0.05 vs vehicle-treated group. B–E, Immunohistochemical localization of PPAR{gamma} in lung of a sham control rat (B), a vehicle-treated rat (C), a ciglitazone-treated rat (D), and a 15d-PGJ2-treated rat (E) 18 h after CLP. Magnification, x100; 1 cm = 78.7 µm. A similar pattern was seen in five or six different tissue sections in each experimental group.

 


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  		FIGURE 4. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on PPAR{gamma} expression in thoracic aortas. Immunohistochemical localization of PPAR{gamma} in the thoracic aorta of a sham control rat (A), a vehicle-treated rat (B), a 15d-PGJ2-treated rat (C), and a ciglitazone-treated rat (D) 18 h after CLP. Magnification, x400; 1 cm = 19.7 µm. A similar pattern was seen in five or six different tissue sections in each experimental group.

 
PPAR{gamma} ligands reduce formation of nitrotyrosine and expression of PARS in thoracic aorta

Overproduction of NO and formation of reactive nitrating species have been proposed to mediate, directly or indirectly via activation of the nuclear enzyme PARS, the vascular dysfunction and hypotension associated with sepsis (19, 20). To further elucidate the effect of PPAR{gamma} ligands on vascular injury, we determined the formation of nitrotyrosine and the expression of PARS. As shown in Fig. 5, thoracic aortas obtained from vehicle-treated rats after 18 h of CLP demonstrated massive immunostaining for nitrotyrosine in endothelium and the smooth muscle layer compared with that in aortas from control sham rats. The formation of nitrotyrosine was associated with the appearance of PARS. However, in vivo treatment with 15d-PGJ2 and ciglitazone markedly reduced nitrosylation of proteins and the expression of PARS (Fig. 5).



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  		FIGURE 5. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on nitrotyrosine (A–D) and PARS immunostaining (E–H) in thoracic aortas 18 h after CLP. Sham rats had no staining for nitrotyrosine (A) or PARS (E). After CLP, a massive dark staining for nitrotyrosine (B) and PARS (F) was localized in the endothelium and smooth muscle layer of vessels from vehicle-treated rats. Immunostaining for nitrotyrosine (C and D) and PARS (G and H) was reduced in rats treated with ciglitazone (C and G) or 15d-PGJ2 (D and H). Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6, and 12 h after CLP. Representative sections (magnification, x400; 1 cm = 19.7 µm) are illustrated. A similar pattern was seen in two or three different tissue sections in each experimental group.

 
Neutrophil infiltration is reduced after treatment with PPAR{gamma} ligands

A serious consequence of sepsis is the occurrence of multiple organ failure, which is preceded by accumulation of neutrophils in major vital organs. Thus, we next evaluated neutrophil infiltration in the lung, colon, and liver by measurement of the activity of myeloperoxidase, an enzyme specific to granulocyte lysosomes, and therefore directly correlated to the number of neutrophils. Myeloperoxidase activity was significantly elevated 18 h after CLP in vehicle-treated rats. Treatment with 15d-PGJ2 or ciglitazone reduced myeloperoxidase activity, suggesting a reduction in neutrophil infiltration (Fig. 6). Treatment with 15d-PGJ2 or ciglitazone did not modify myeloperoxidase activity in control rats (i.e., not subjected to CLP; data not shown).



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  		FIGURE 6. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on myeloperoxidase (MPO) activity in lung (A), liver (B), and colon (C) of rats 18 h after CLP. Each data point represents the mean ± SEM of six to 11 animals for each group. *, p < 0.05 vs sham rats; #, p < 0.05 vs vehicle-treated rats. Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6, and 12 h after CLP.

 
PPAR{gamma} ligands reduce production of TNF-{alpha}, IL-6, and IL-10

A hallmark of sepsis is the overproduction of cytokines. A substantial increase in TNF-{alpha}, IL-6, and IL-10 production was found 18 h after CLP in vehicle-treated rats. Treatment with 15d-PGJ2 or ciglitazone reduced cytokine production (Fig. 7). Treatment with 15d-PGJ2 or ciglitazone did not modify basal levels of cytokines in control rats (i.e., not subjected to CLP; data not shown).



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  		FIGURE 7. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on plasma levels of TNF-{alpha} (A), IL-6 (B), and IL-10 (C) in rats 18 h after CLP. Each data point represents the mean ± SEM of six animals for each group. *, p < 0.05 vs sham rats; #, p < 0.05 vs vehicle-treated rats. Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6, and 12 h after CLP.

 
PPAR{gamma} ligands reduce activation of NF-{kappa}B, degradation of I{kappa}B{alpha}, and activity of IKK

To investigate the cellular mechanisms by which PPAR{gamma} ligands may attenuate the inflammatory response during sepsis, we evaluated the nuclear activation of NF-{kappa}B, a major transcription factor involved in the signal transduction of sepsis (3, 4). In the lungs of vehicle-treated rats, polymicrobial sepsis resulted in the early activation of NF-{kappa}B, with activity reaching a maximum within 3–6 h after CLP. In contrast, treatment with 15d-PGJ2 or ciglitazone significantly reduced the DNA binding activity of NF-{kappa}B (Fig. 8).



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  		FIGURE 8. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on NF-{kappa} B DNA binding in lung. A, Representative autoradiograph of EMSA for NF-{kappa}B; B, image analysis of activation of NF-{kappa} B determined by densitometry. The fold increase was calculated vs the respective sham value (time zero), which was set at 1.0. Results are representative of three separate time-course experiments. *, p < 0.05 vs vehicle-treated rats. Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6, and 12 h after CLP.

 
Because degradation of I{kappa}B{alpha} by a phosphorylation- and ubiquitination-dependent pathway represents an important event for NF-{kappa}B nuclear translocation and the initiation of transcription (3, 4), we further determined the degradation of I{kappa}B{alpha} and the activity of IKK. In vehicle-treated rats, the cytosol content of I{kappa}B{alpha} was reduced in a time-dependent manner after induction of polymicrobial sepsis, thus suggesting the occurrence of degradation (Fig. 9). These events were preceded by the early activation of IKK in the lung, with activity reaching a maximum after 3 h (Fig. 10). In contrast, in 15d-PGJ2-treated or ciglitazone-treated rats, the I{kappa}B{alpha} content was significantly higher than that in vehicle-treated rats, suggesting inhibition of degradation (Fig. 9). The phosphorylative activity of IKK was also significantly reduced in 15d-PGJ2-treated or ciglitazone-treated rats compared with that in vehicle-treated rats (Fig. 10).



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  		FIGURE 9. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on cytosolic content of I{kappa}B{alpha}. A, Representative Western blot analysis for I{kappa}B{alpha}; B, image analysis of I{kappa}B{alpha} determined by densitometry. The fold increase was calculated vs the respective sham value (time zero), which was set at 1.0. Results are representative of three separate time-course experiments. *, p < 0.05 vs vehicle-treated rats. Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6, and 12 h after CLP.

 


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  		FIGURE 10. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on activation of IKK. A, Representative analysis for IKK activity; B, image analysis of enzymatic activity determined by densitometry. IKK activity was estimated as the ability to phosphorylate GST-I{kappa}B{alpha} after immunoprecipitation of proteins with specific anti-IKK{gamma} Ab. The fold increase was calculated vs the respective sham value (time zero), which was set at 1.0. Results are representative of three separate time-course experiments. *, p < 0.05 vs vehicle-treated rats. Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6, and 12 h after CLP.

 
PPAR{gamma} ligands reduce activation of AP-1 and JNK activity

Because activation of AP-1 has also been implicated in sepsis, we further determined the nuclear activation of this factor. AP-1 activity steadily increased after CLP, with maximum DNA binding at 6 h (Fig. 11).



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  		FIGURE 11. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on AP-1 DNA binding in lung. A, Representative autoradiograph of EMSA for AP-1; B, image analysis of activation of AP-1 determined by densitometry. The fold increase was calculated vs the respective sham value (time zero), which was set at 1.0. Results are representative of three separate time-course experiments. *, p < 0.05 vs vehicle-treated rats. Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6, and 12 h after CLP.

 
Because phosphorylation of the components of AP-1, such as c-Jun, by JNK represents an important event for the stability and activation of the transcription factor (5), we further determined the nuclear activity of JNK. A time-course study showed that JNK activity increased as early as 3 h after the induction of polymicrobial sepsis and declined thereafter in vehicle-treated rats (Fig. 11). However, in vivo treatment with the PPAR{gamma} ligands, ciglitazone and 15d-PGJ2, markedly reduced the degree of activity of JNK and AP-1 (Figs. 11 and 12).



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  		FIGURE 12. Effect of in vivo treatment with 15d-PGJ2 or ciglitazone on activation of JNK. A, Representative analysis for JNK activity; B, image analysis of enzymatic activity determined by densitometry. JNK activity was estimated as the ability to phosphorylate GST-c-Jun after immunoprecipitation of proteins with specific anti-JNK1 Ab. The fold increase was calculated vs the respective sham value (time zero), which was set at 1.0. Results are representative of three separate time-course experiments. *, p < 0.05 vs vehicle-treated rats. Treated rats received 15d-PGJ2 (1 mg/kg) or ciglitazone (5 mg/kg) by i.p. injection 1, 6,and 12 h after CLP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The present study demonstrates the therapeutic potential of PPAR{gamma} ligands in a clinically relevant model of sepsis. Specifically, we demonstrate that treatment with 15d-PGJ2 and ciglitazone ameliorates hypotension and vascular injury and reduces neutrophil infiltration in lung, colon, and liver and the elevation of cytokines in plasma. The reduction of the inflammatory process is also associated with a significant improvement in survival. Furthermore, our data provide evidence that the beneficial effects of the PPAR{gamma} ligands are secondary to a negative modulation of IKK/NF-{kappa}B and JNK/AP-1 signal transduction pathways.

PPAR{gamma} expression is altered during sepsis

PPAR{gamma} is constitutively expressed in several cell types, including adipocytes, endothelial cells, smooth muscle cells, epithelial cells, macrophages, and related cells of the immune system (6, 11, 21). In our study we observed that during polymicrobial sepsis, the expression of PPAR{gamma} is altered in the lung and in the vasculature. We demonstrated that PPAR{gamma} expression was down-regulated in the bronchial epithelium and in smooth muscle and endothelial cells of the thoracic aorta. Interestingly, the expression of PPAR{gamma} decreased in a time-dependent fashion during sepsis. This temporal decrease correlated well with the occurrence of hypotension and the severity of the inflammatory response. Treatment with the PPAR{gamma} ligands caused up-regulation of PPAR{gamma} to normal constitutive expression. Previous studies have also suggested that PPAR{gamma} expression may be altered in an inflammatory process. In adipose tissue, mRNA and protein expression decreased after mice were challenged in vivo with endotoxin (22). In other studies, inflammatory cytokines, including TNF-{alpha}, IL-1{beta}, and IL-6 also decreased PPAR{gamma} expression in adipose tissue. The suppression of PPAR{gamma} mRNA expression caused by the cytokines was reversed by treatment with troglitazone and pioglitazone (23). Similarly, Hinz and colleagues (24) found that stimulation of monocytes with endotoxin resulted in an 88% inhibition of PPAR{gamma} mRNA expression that was fully restored by 15d-PGJ2, but only partially restored by ciglitazone. Taken together, these findings suggest that the reduction of the endogenous activity of PPAR{gamma} may contribute to the overwhelming systemic inflammatory response of sepsis. Furthermore, our results indicate that the anti-inflammatory properties of 15d-PGJ2 and ciglitazone may extend to the regulation of nuclear expression of PPAR{gamma} in target cells. Several mechanisms by which treatment of PPAR{gamma} ligands restores reduced levels of PPAR{gamma} in sepsis may be hypothesized. Mitogen-activated kinases, such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), have been proposed to induce the suppression of PPAR{gamma} transcriptional activation through phosphorylation of the receptor (25, 26, 27). In previous in vitro studies in immunostimulated macrophages, we have demonstrated that 15d-PGJ2 suppresses ERK1/2 activation (28), which may be in part a mechanism for maintaining PPAR{gamma} expression and activation. Furthermore, Shibuya and colleagues (29) have reported that PPAR{gamma} is nitrated in cells stimulated with endotoxin or TNF-{alpha}, and this nitration affects the receptor’s ability to translocate into the nucleus. Although the reactions leading to nitrotyrosine formation are still under debate (30), nitration of tyrosine residues in proteins has been observed in septic shock and has been suggested to interfere with protein function and signal transduction (31, 32). Several reports, including our previous studies, have shown that the production of nitrogen reactive species and the formation of nitrotyrosine occur in both endothelial and vascular smooth muscle cells after inflammatory challenge (33, 34, 35, 36). Electron microscopic examination of cultured rat aortic smooth muscle cells indicated that nitrated proteins are localized in the endoplasmic reticulum, mitochondria, and the cell nucleus. The localization of nitrated proteins in the nucleus implies that specific proteins are targeted for nitration (36). In our experiments we found that nitrotyrosine formation in thoracic aorta was associated with down-regulation of PPAR{gamma}. The formation of nitrotyrosine was reversed by in vivo treatment with 15d-PGJ2 or ciglitazone. Thus, the ability of PPAR{gamma} ligands to suppress nitrotyrosine formation may be another mechanism by which PPAR{gamma} ligands maintain PPAR{gamma} expression in vessels of rats subjected to polymicrobial sepsis. However, it is difficult to establish from our in vivo data whether down-regulation of PPAR{gamma} correlates with decreased PPAR{gamma} function. Further research is required to examine the changes in PPAR{gamma} activity during sepsis.

Other studies have reported that PPAR{gamma} may also be up-regulated in immune cells. For example, studies by Leininger and colleagues (37) demonstrate that in porcine white blood cells, PPAR{gamma} protein expression increased 2-fold over the basal level in response to acute endotoxemia. Increased activity of PPAR{gamma} has been demonstrated in macrophages stimulated with a subthreshold concentration of endotoxin and IFN-{gamma}, which induces cell desensitization (38). Taken together with our data, these studies suggest that PPAR{gamma} expression may be differently regulated by microbial pathogens and may depend on the cell immunological function and specificity.

Endogenous arachidonic acid metabolites and, more specifically, the PGJ2 metabolites have been shown to be natural PPAR{gamma} agonists (10, 11). In our study we did not measure the production of 15d-PGJ2 or other endogenous cyclopentenone PGs. Increased activity of COX-2 and endogenous synthesis of eicosanoids in response to endotoxemia and sepsis occur in a variety of animal species and in humans (39). Whether these changes also correlate with changes in the production of 15d-PGJ2 metabolites remains to be elucidated. However, it has been proposed that during inflammation, levels of endogenous 15d-PGJ2 correlate with changes in the severity of the inflammatory response. It has been reported that an early induction of COX-2 coincided with production of PGE2 in rats subjected to carrageenan-induced pleurisy. By contrast, PGD2 and 15d-PGJ2 declined as inflammation increased. However, at 48 h there was a second induction of COX-2 with minimal PGE2 synthesis, but with increased PGD2 and 15d-PGJ2. COX-2 inhibitors attenuated the early inflammation, but augmented the inflammation in response to the delayed COX-2 expression. The latter effect was reversed by replacement of PGD2 and 15d-PGJ2 (40). The possibility that endogenous production of 15d-PGJ2 may change the course of inflammation may also explain the different and conflicting effects of COX-2 inhibitors in sepsis (41, 42). Taken together with our findings, these reports suggest that the use of cyclopentenone derivatives may represent a novel therapeutic approach to the treatment of inflammation.

PPAR{gamma} ligands alter signal transduction mediated by NF-{kappa}B and AP-1

The anti-inflammatory mechanisms of PPAR{gamma} ligands have not yet established in vivo. In our study we demonstrated that regulation of signal transduction mechanisms accounts for the anti-inflammatory role of PPAR{gamma} during sepsis. Numerous experimental studies have proven that activation of both NF-{kappa}B and AP-1 is implicated in sepsis. Enhancement of AP-1 and NF-{kappa}B DNA binding activity has been found in skeletal muscle and liver in rats subjected to CLP (43, 44). With particular clinical relevance, NF-{kappa}B binding activity has been found to be increased in patients with acute inflammation and sepsis and to be correlated with clinical severity and mortality (45). NF-{kappa}B activation increased markedly before death in mononuclear leukocytes of patients with systemic inflammatory response syndrome (46). These findings strongly suggest that modulation of NF-{kappa}B and AP-1 may represent a therapeutic target for the treatment of sepsis and septic shock. In our study we found that in vivo treatment with 15d-PGJ2 or ciglitazone markedly decreased the nuclear activation of both NF-{kappa}B and AP-1 activity in the lung. When examined at a more molecular level, the reduction of the DNA binding of these transcription factors appeared to be a downstream event of reduced activity of their regulatory kinases IKK and JNK.

The present study is in agreement with other reports that ascribe to PPAR{gamma} a regulatory role on transcription and gene expression. The suppression of NF-{kappa}B stimulation has been demonstrated to mediate the antiatherosclerotic effect of troglitazone in obese patients (47). Studies from our laboratory have shown that 15d-PGJ2 and troglitazone may affect signaling to ERK1/2 and induce I{kappa}B{alpha} synthesis in in vitro macrophages stimulated by endotoxin or heat-killed Staphylococcus aureus (28).

However, it is difficult to establish from our in vivo data whether the modulation of signal transduction is mediated via a direct interaction of the ligands with the nuclear PPAR{gamma} or via other PPAR{gamma}-independent mechanisms. It has been proposed that PPAR{gamma} may modulate the expression of an inflammatory gene by direct transrepression. PPAR{gamma} inhibited iNOS expression by direct interaction with CREB-binding protein, thus limiting its availability for NF-{kappa}B and AP-1 transcription (48). In human vascular endothelial cells a direct inhibition of PPAR{gamma} on AP-1 binding has been accounted for the inhibition of endothelin-1 gene expression (49). In contrast, several studies have demonstrated that synthetic and natural PPAR{gamma} ligands have PPAR{gamma}-independent effects. In microglial cells 15d-PGJ2 inhibited NF-{kappa}B transcriptional activity and inhibited iNOS mRNA and NO production, whereas the PPAR{gamma} agonist troglitazone had no effect (50). Similarly, exposure of rat cardiac myocytes to 15d-PGJ2, but not to rosiglitazone, resulted in up-regulation of the expression of heme-oxygenase-1 mRNA (51). In another study Thieringer and colleagues (52) demonstrated that only 15d-PGJ2, of five PPAR{gamma} agonists examined, inhibited LPS-induced TNF-{alpha} and IL-6 production in vitro and in vivo over a wide range of concentrations. The NF-{kappa}B pathway appears to be a target for PPAR{gamma}-independent effects of cyclopentenone PGs. For example, 15d-PGJ2 inhibited endotoxin-induced degradation of I{kappa}B{alpha} and NF-{kappa}B gene activation by covalent modification of critical cysteine residues in IKK and the DNA binding domain of the p65 NF-{kappa}B subunit (53). In PPAR{gamma}-deficient cells 15d-PGJ2 was able to modify cysteine residues of IKK and inhibit the activation of NF-{kappa}B (54). More recently, 15d-PGJ2 has been demonstrated to covalently modify cysteine 62 of NF-{kappa}B subunit p50 and to reduce recombinant DNA binding in a dose-dependent manner (55). Other mechanisms have been proposed for the regulatory role of PPAR{gamma} ligands in inflammation. It has been shown that the ability of 15d-PGJ2 and troglitazone to block cytokine-induced iNOS in macrophages and IL-1-induced I{kappa}B{alpha} degradation and JNK phosphorylation in islet cells was associated with the expression of heat shock protein 70, which is known to have broad cytoprotective properties (56).

Therefore, it is conceivable that PPAR{gamma} ligands may have several cellular targets in vivo. In our study both the thiazolidinedione ciglitazone and the cyclopentenone 15d-PGJ2 exerted similar inhibitory effects. Therefore, we hypothesize that these ligands may affect transcription, probably through direct activation of PPAR{gamma}. This hypothesis is corroborated by the fact that after treatment with the ligands PPAR{gamma} expression was up-regulated in cellular nuclei, where transcription factors and JNK are present in an active form during sepsis. In contrast, these ligands may also affect transcription through direct inhibition of transcription factors and/or kinases.

PPAR{gamma} ligands blunt the inflammatory response

Acute inflammatory response and early cellular infiltration are the first line of host defense to facilitate pathogen clearance. Specifically, neutrophils and lymphocytes play a central role in the immune and wound-healing processes of sepsis. However, during sepsis and septic shock, this defense mechanism becomes exaggerated and leads to organ damage and failure (1, 57, 58). For example, a vigorous activation of neutrophils can have deleterious effects as neutrophils accumulate in tissues and release reactive oxygen species and proteases that injure host structures (57, 58). In contrast, widespread lymphocyte depletion by apoptosis may contribute to the lymphocytopenia observed in patients with sepsis and to decreased elimination of bacteria and bacterial products in sepsis (59). In our experiments we found that treatment with PPAR{gamma} ligand was able to counteract the inflammatory process without altering the bacterial clearance. However, further investigation is needed to ascertain whether attenuation of neutrophilia and/or lymphocytopenia may contribute to the anti-inflammatory effects of PPAR{gamma} ligands. Nevertheless, these data suggest that PPAR{gamma} ligands may represent a valid adjunctive therapy to antibiotics.

In vitro reports have suggested that NF-{kappa}B and/or AP-1 pathways control several inflammatory genes, such as iNOS, adhesion molecules, and cytokines (3, 4, 5, 60, 61). In our study PPAR{gamma} ligands were able to blunt the plasma release of several cytokines, such as IL-6, IL-10, and TNF-{alpha}, and to reduce neutrophil infiltration in major vital organs, thus suggesting a negative modulation of gene expression of cytokines and adhesion molecules. Furthermore, enhanced production of NO has been shown to contribute to the hypotension and vascular hyporeactivity to various constrictor agents in septic shock (62). NO, directly or indirectly through formation of peroxynitrite, produces cellular injury and death via several mechanisms, including peroxidation of membrane lipids, protein nitration and nitrosylation, and DNA damage (19, 20, 63). The occurrence of DNA breaks has been shown to activate the nuclear enzyme PARS, resulting in depletion of the cellular energy substrates NAD and ATP. This process, termed PARS suicide, has been proposed to play an important role in inflammation and shock (19, 20, 64). In the present study the beneficial hemodynamic effects of the PPAR{gamma} ligands in septic animals were associated with inhibition of formation of nitrotyrosine and the expression of PARS in aortas, thus suggesting a reduction in the production of nitrogen-reactive species.

Because a critical function of PPAR{gamma} involves glucose metabolism, one can expect that treatment with PPAR{gamma} ligands would result in modification of blood glucose levels. In this regard, thiazolidinediones have been widely used as antihyperglycemic drugs for the treatment of insulin-resistant diabetes (65). In our study blood glucose levels were carefully monitored to avoid hypoglycemia after treatment with PPAR{gamma} ligands. However, we found that 15d-PGJ2 or ciglitazone did not induce hypoglycemia. It is also important to mention that the clinical consequences of sepsis are increased levels of gluconeogenic hormones, altered transport of glucose, and insulin resistance (66). The burst of endogenous catecholamines and the therapeutic use of exogenous catecholamines to maintain hemodynamic function can also contribute to glucose dysmetabolism in septic patients. In our experiments one rat of six indeed exhibited an increase in glucose over normal levels. Taken together, these data suggest that 15d-PGJ2 and ciglitazone may also exert a beneficial effect in maintaining normal glucose levels.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
The present study demonstrated that PPAR{gamma} ligands, ciglitazone and 15d-PGJ2, exhibit potent anti-inflammatory properties in experimental sepsis. This beneficial effect appears to be secondary to the regulation of both NF-{kappa}B and AP-1 signaling pathways. However, further investigation is warranted to investigate PPAR{gamma}-dependent and -independent effects of these agents. Because many PPAR{gamma} ligands are already in clinical application, these studies hold the promise of new therapeutic strategies to treat sepsis.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R01HL60730 and R01 GM-67202 (to B.Z.) and R01GM27673 (to J.A.C.). Back

2 Address correspondence and reprint requests to Dr. Basilia Zingarelli, Division of Critical Care Medicine, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: basilia.zingarelli{at}cchmc.org Back

3 Abbreviations used in this paper: IKK, I{kappa}B kinase complex; CLP, cecal ligation and puncture; COX-2, cyclooxygenase 2; ERK1/2, extracellular signal-regulated kinases 1 and 2; I{kappa}B{alpha}, inhibitor {kappa}B{alpha}; JNK, c-Jun NH2-terminal kinase; PARS, poly(ADP-ribose) synthetase; PGJ2, 15-deoxy-{Delta}12,14-PGJ2; PPAR{gamma}, peroxisome proliferator activator receptor-{gamma}; 15d-iNOS, inducible NO synthase. Back

Received for publication May 29, 2003. Accepted for publication October 2, 2003.


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