Role of PPAR-δ in the development of zymosan-induced multiple organ failure: an experiment mice study

Male CD mice (20-22 g; Charles River; Milan; Italy) were housed in a controlled environment and provided with standard rodent chow and water. The study was approved by the University of Messina Review Board for the care of animals. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M.116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986)

Mice were randomly allocated into the following groups: (1) Zymosan + vehicle group. Mice were treated intraperitoneally (i.p.) with zymosan (500 mg/kg, suspended in saline solution, i.p.) and with the vehicle for GW0742 (10% dimethylsulfoxide (DMSO) (v/v) i.p), 1 and 6 h after zymosan administration, n = 10; (2) Zymosan + GW0742 group. Identical to the Zymosan + vehicle group but were administered GW0742 (0,3 mg/kg 10% DMSO i.p) at 1 and 6 hour after zymosan instead of vehicle, n = 10; (3) Sham + vehicle group. Identical to the Zymosan + vehicle group, except for the administration of saline instead of zymosan, n = 10; (4) Sham + GW0742 group. Identical to Sham + vehicle group, except for the administration of GW0742 (0,3 mg/kg in 10% DMSO i.p) 1 and 6 hour after saline administration, n = 10. Eighteen hours after administration of zymosan, animals were assessed for shock as described below. In another set of experiments, animals (n = 30 for each group) were randomly divided as described above and monitored for loss of body weight and mortality for 7 days after zymosan or saline administration.

Clinical severity of systemic toxicity in the mice was scored during the experimental period, (7 days) after zymosan or saline injection, on a subjective scale ranging from 0 to 3; 0 = absence, 1 = mild, 2 = moderate, 3 = serious. The scale was used for each of the toxic signs (conjunctivitis, ruffled fur, diarrhea and lethargy) observed in the animals. The final score was produced upon totaling each evaluation (maximum value 12). All clinical score measurements were performed by an independent investigator, who had no knowledge of the treatment received by each respective animal.

Eighteen hours after zymosan or saline injection, all animals (n = 10 for each group) were killed under ether anesthesia in order to evaluate the development of acute inflammation in the peritoneum. Through an incision in the linea alba, 5 ml of phosphate buffered saline (PBS, composition in mM: NaCl 137, KCl 2.7, NaH₂PO₄ 1.4, Na₂HPO₄ 4.3, pH 7.4) was injected into the abdominal cavity. Washing buffer was removed with a plastic pipette and was transferred into a 10 ml centrifuge tube. The amount of exudate was calculated by subtracting the volume injected (5 ml) from the total volume recovered. Peritoneal exudate was centrifuged at 7000 × g for 10 min at room temperature.

At 18 h after treatment, the mice were anesthetized with intramuscular injection of ketamine/xylazine. The mice were injected with 5 mL of ice-cold RPMI-1640 medium (Gibco Inc., Grand Island, NY) with 10% heparin (50 U.I./ml), into the abdominal cavity. The peritoneal cavities were massaged for 1 min and the lavage fluid was collected. Peritoneal exudates cell (PEC) counts were carried out in a hemocytometer by mixing 100 μL of peritoneal cell exudate and 100 μL of eosin. The PEC was spin in a cytocentrifuge at 50 × g for 5 min onto a slide for the differential count. The slides were carefully removed and allowed to air dry briefly. PEC cytospins were stained with Wright-Giemsa stain. PEC cytospins were also stained with neutrophil/mast cell-specific chloroacetate esterase staining and macrophage/monocyte-specific alpha naphthyl butyrate esterase stains for the differential count.

Nitrite/nitrate (NO₂/NO₃) production, an indicator of NO synthesis, was measured in plasma and in the exudate samples collected 18 hours after zymosan or saline administration, as previously described [23, 25]. Nitrate concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrate prepared in saline solution.

Tyrosine nitration and PARP activation were detected, as previously described [26], in lung, liver and intestine sections using immunohistochemistry. At 18 hours after zymosan or saline injection, tissues were fixed in 10% (w/v) PBS-buffered formalin and 8 μm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin (Vector Laboratories, Burlingame, CA). The sections were then incubated overnight with 1:1000 dilution of primary anti-nitrotyrosine antibody (Millipore, 1:500 in PBS, v/v), anti-poly(ADP)-ribose (PAR) antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v), purified hamster anti-mouse ICAM-1 (CD54) (1:500 in PBS, w/v) (DBA, Milan, Italy), purified goat polyclonal antibody directed towards P-selectin which reacts with mice, anti-Bax rabbit polyclonal antibody (1:500 in PBS, v/v), anti-Bcl-2 polyclonal antibody rat (1:500 in PBS, v/v), anti-TNF-α antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v), anti-IL-1β antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v), or anti-Fas Ligand antibody (Abcam,1:500 in PBS, v/v). Controls included buffer alone or non-specific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated specific secondary anti-IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA). To verify the binding specificity for nitrotyrosine, PARP, ICAM-1, P-Selectin, Bax, Bcl-2, TNF-α and IL-1β and FasL, some sections were also incubated with primary antibody only (no secondary antibody) or with secondary antibody only (no primary antibody). In these situations, no positive staining was found in the sections indicating that the immunoreactions were positive in all the experiments carried out. In order to confirm that the immunoreactions for the nitrotyrosine were specific some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity.

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling assay (TUNEL) was conducted by using a TUNEL detection kit according to the manufacturer's instruction (Apotag horseradish peroxidase kit; DBA, Milan, Italy). Briefly, sections were incubated with 15 2 g/Ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H₂O₂ for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyl transferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated deoxyuridine 5-triphosphate in TdT buffer, incubated in a humid atmosphere at 37-C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-fluorescein isothiocyanate horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.

Tissues were homogenized in cold lysis buffer A (HEPES 10 mM pH = 7.9; KCl 10 mM;EDTA 0.1 mM; EGTA 0.1 mM; DTT 1 mM; PMSF 0.5 mM; Trypsin inhibitor 15 μg/ml; PepstatinA 3 μg/ml; Leupeptin 2 μg/ml; Benzamidina 40 μM). Homogenates were centrifuged at 12000 g for 3 min at 4°C, and the supernatant (cytosol + membrane extract) was collected to evaluate contents of iNOS, IkB-α, Bax, Bcl-2 and β-actin. The pellet was resuspended in buffer C (HEPES 20 mM; MgCl₂ 1.5 mM; NaCl 0.4 mM; EDTA 1 mM; EGTA 1 mM; DTT 1 mM; PMSF 0.5 μg/ml; Leupeptin 2 μg/ml; Benzamidina 40 μM; NONIDET P40 1%; Glicerolo 20%) and centrifuged at 12000 g for 12 min at 4°C, and the supernatant (nuclear extract) was collected to evaluate the content of NF-kB p65 and LaminB1. Protein concentration in the homogenate was determined by Bio-Rad Protein Assay (BioRad, Richmond CA) and 50 μg of cytosol and nuclear extract from each sample was analysed. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred on a PVDF membrane (Hybond-P Nitrocellulose, Amsherman Biosciences, UK). The membrane was blocked with 0.1% TBS-Tween containing 5% non fat milk for 1 h at room temperature. After the blocking, the membranes were incubated with the relative primary antibody overnight at 4°C; anti-iNOS TYPE II diluted 1:1000 (Transduction Laboratories), anti-IkB-α diluted 1:1000, anti-Bax diluted 1:500, anti-Bcl2 diluted 1:1000, anti-NFkB p65 diluted 1:250, anti-β-actin 1:5000 (Santa Cruz Biotechnology, CA) and anti-Laminin B1. After the incubation, the membranes were washed three times for ten minutes with 0.1% TBS Tween and were then incubated for one hour with peroxidase-conjugated anti- mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, USA) diluted 1:2000, the membranes were then washed three times for ten minutes and protein bands were detected with SuperSignal West Pico Chemioluminescent (PIERCE). Densitometric analysis was performed with a quantitative imaging system (ImageJ).

The levels of TNF and IL-1β were evaluated in the plasma at 18 hours after zymosan or saline administration. The assay was conducted using a colorimetric commercial kit (Calbiochem-Novabiochem, La Jolla, CA). The ELISA has a lower detection limit of 10 pg/ml.

Myeloperoxidase (MPO) activity, which was used as an indicator of PMN infiltration into the lung and intestinal tissues, was measured as previously described [27].

Blood samples were taken at 18 h after zymosan or saline injection and centrifuged (1610 × g for 3 min at room temperature) to separate plasma. Levels of amylase, lipase, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubine and alkaline phosphatase were measured by a veterinary clinical laboratory using standard laboratory techniques. For the evaluation of acid base balance and blood gas analysis (indicator of lung injury) arterial blood levels of pH, PaO₂ and PaCO₂ and bicarbonate ion (HCO₃^-) were determined by pH/Blood gases Analyser as previously described [28].

Lung, liver and small intestine samples were taken 18 hours after zymosan or saline injection. The tissue slices were fixed in Dietric solution [14.25% (v/v) ethanol, 1.85% (w/v) formaldehyde, 1% (v/v) acetic acid] for 1 week at room temperature, dehydrated by graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, New Jersey, USA). Sections (thickness 7 μm) were deparaffinized with xylene, stained with hematoxylin and eosin and observed in Dialux 22 Leitz microscope.

Unless stated otherwise, all reagents and compounds were obtained from Sigma Chemical Company (Milan, Italy).

All values in the figures and text are expressed as mean ± standard error of the mean (s.e.m.) of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments (histological or immunohistochemistry coloration) performed on different experimental days on the tissue sections collected from all animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni's post-hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. Statistical analysis for survival data was calculated by Kaplan-Meier survival analysis The Mann-Whitney U test (two-tailed, independent) was used to compare medians between the body weight and the clinical score. For such analyses, p < 0.05 was considered significant.

At 18 h after zymosan administration, histological evaluation of pancreas (Figure 1D) lung (Figure 1E) and gut (Figure 1F) sections demonstrated several marked pathological changes. In the pancreas, there was extravasation of neutrophils (Figure 1D). Lung biopsy revealed inflammatory infiltration by neutrophils, macrophages and plasma cells (Figure 1E). In the gut, there was infiltration of inflammatory cells, edema in the space bounded by the villus, and separation of the epithelium from the basement membrane (Figure 1F). Treatment with GW0742 markedly reduced the histological damage in the pancreatic (Figure 1G), pulmonary (Figure 1H) and intestinal (Figure 1I) tissue. No histological alteration was observed in the pancreas (Figure 1A), lung (Figure 1B) or gut (Figure 1C) from sham-treated mice.

Administration of zymosan caused severe illness in the mice, characterized by systemic toxicity and significant loss of body weight (Figure 2A, 1B). At the end of the observation period (7 days), 75% of zymosan-treated mice were dead (Figure 2C). Treatment with GW0742 reduced the development of systemic toxicity (Figure 2A), loss in body weight (Figure 2B) and mortality (Figure 2C), caused by zymosan. GW0742 treatment did not cause any significant changes in these parameters in sham mice (Figure 2A, 2B, 2C).

The development of acute peritonitis occurred 18 h after zymosan administration was indicated by the production of turbid exudates (Figure 3A). The total number of peritoneal exudate cells (PEC) (Figure 3B) was determined by trypan blue staining following intraperitoneal administration of zymosan or saline solution. This demonstrated a significant increase in the polymorphonuclear leukocyte number when compared with sham mice, which demonstrated no abnormalities in the peritoneal cavity or fluid.

Zymosan injection in mice was associated with an increase in PEC counts at 18 h, when compared to the saline controls (Figure 3B). Since there was a quantitative increase in PECs following zymosan injection, cytospin preparations were performed of the PEC for a differential estimation of the types of cells present. Wright-Giemsa stained slides of all controls appeared to contain mostly mononuclear cells including resident macrophages and lymphocytes and very few polymorphonuclear neutrophils, as previously demonstrated [29]. All cells appeared healthy and intact. At 18 h after zymosan administration, almost all cells appeared lysed, and because of excessive phagocytosis by the leukocytes, the neutrophils could not be differentiated from macrophages. Since the cells appeared lysed and the nucleus could not be differentiated, cell staining for specific esterases for neutrophil and macrophages were carried out in order to attempt differentiation between cell populations in the zymosan treated animals. In agreement with previous observations [29], we confirmed the presence of 90% mononuclear cells in the peritoneal cavity along with 10% PMNs in all the sham-treated animals. In contrast, the zymosan-treated samples could not be differentiated due to excessive phagocytosis and lysis of cells. Exudate formation (Figure 3A) and the degree of PEC count (Figure 3B) were significantly reduced in mice treated with GW0742.

To investigate the inflammatory cellular mechanisms by which treatment with GW0742 may attenuate the development of zymosan-induced injury, we evaluated IκB-α degradation and nuclear NF-κB p65 translocation by Western Blot analysis. A basal level of IκB-α was detected in the lung tissues of sham-animals (Figure 4a, see densitometric analysis Figure 4a1), whereas in zymosan-treated mice, IκB-α levels were substantially reduced (Figure 4a, see densitometric analysis Figure 4a1). GW0742 prevented zymosan-induced IκB-α degradation, with IκB-α levels observed in these animals similar to those of the sham group (Figure 4a, see densitometric analysis Figure 4a1). In addition, zymosan administration caused a significant increase in NF-kB p65 levels in the nuclear fractions from lung tissues, compared to the sham-treated mice (Figure 4b, see densitometric analysis Figure 4b1). GW0742 treatment significantly reduced the levels of NF-kB p65 in the lung (Figure 4a, see densitometric analysis Figure 4a1).

The modulation of GW0742 on the inflammatory process through the regulation of cytokine secretion was assessed by determination of plasmatic levels of the pro-inflammatory cytokines TNF-α and IL-1β. A substantial increase in TNF-α and IL-1β formation was observed in zymosan-treated mice when compared to sham mice (Figure 5A, 5B, respectively), while a significant inhibition of TNF-α and IL-1β was observed when animals with zymosan-induced injury were treated with GW0742 (Figure 5B, respectively). In addition, tissue sections of pancreas, lung and gut obtained from animals 18 h after zymosan administration, demonstrated positive staining for TNF-α and IL-1-β in pancreas, (Figure 6A, 7A respectively) lung (Figure 6B, 7B respectively) and gut(Figure 6C, 7C respectively) On the contrary the staining for TNF-α and IL-1-β was visibly and significantly reduced in zymosan mice treated with GW0742 in pancreas, (Figure 6D, 7D respectively) lung (Figure 6E, 7E respectively) and gut (Figure 6F, 7F respectively). In the pancreas, lungs and gut of sham animals no positive staining was observed for TNF-α (data not shown) or IL-1-β (data not shown).

At 18 h after zymosan administration, expression of the adhesion molecules ICAM-1 and P-selectin were evaluated to assess neutrophil infiltration. In zymosan-treated mice, an increase of immunohistochemical staining for ICAM-1 and P-selectin was demonstrated in the pancreas (Figure 8A, 9A respectively) lung (Figure 8B, 9B respectively) and gut(Figure 8C, 9C respectively), (see arrows) while the immunostainings for ICAM-1 and P-selectin were markedly reduced in pancreas (Figure 8D, 9D respectively) lung (Figure 8E, 9E respectively) and gut(Figure 8F, 9F respectively) tissues obtained from mice, that were treated with GW0742. No staining for either ICAM-1 or P-selectin was found in tissue sections obtained from sham-treated mice (data not shown).

The accumulation of neutrophils in the intestine and lung is a hallmark of multiple organ failure induced by zymosan. An indirect assessment of neutrophil infiltration was carried out by measuring the activity of myeloperoxidase (MPO), an enzyme that is contained in (and specific for) PMN lysosome dysfunction [23, 25]. At 18 h after zymosan administration, MPO activity was significantly increased in the lungs (Figure 10A) and gut (Figure 10B) of zymosan-challenged mice, when compared with sham-operated mice (Figure 10A, B). MPO activity was markedly reduced in the lungs (Figure 10A) and gut (Figure 10B) of zymosan-challenged mice treated with GW0742.

The analysis of exudates (Figure 11A) and plasma (Figure 11B) levels validate the biochemical and inflammatory changes observed in the peritoneal cavity of zymosan-treated mice, showing a significant increase of nitrite/nitrate (NOx) concentration (Figure 11A, 11B, respectively) when compared with sham-operated mice. These values were significantly reduced in mice treated with GW0742 (Figure 11A, 11B, respectively). We assessed iNOS expression in samples of pulmonary tissue by Western Blot analysis (Figure 11Ca, see densitometric analysis a1). A significant increase in iNOS expression has been demonstrated in samples of lung obtained from zymosan-injected mice, when compared with sham-operated mice (Figure 11Ca, see densitometric analysis a1). In contrast, a significant decrease in iNOS expression was clearly observed in the zymosan-challenged mice treated with GW0742 (Figure 11Ca, see densitometric analysis a1).

To determine the localization of "peroxynitrite formation" and/or other nitrogen derivatives produced during multiple organ failure, zymosan-induced nitrotyrosine, a specific marker of nitrosative stress, was measured by immunohistochemical analysis in sections of pancreas, lung and gut tissues, using a specific anti-nitrotyrosine antibody. The samples obtained from sham-operated mice did not stain for nitrotyrosine (data not shown), while sections from zymosan-induced mice exhibited positive staining for nitrotyrosine in pancreas (Figure 12A), lung (Figure 12B) and gut (Figure 12C) tissues (see arrows). A marked reduction in nitrotyrosine staining was found in the pancreas (Figure 12D), lung (Figure 12E) and gut (Figure 12F) of the zymosan-challenged mice treated with GW0742.

Sections of pancreas, lung and gut were taken at 18 h after zymosan administration in order to determine the activation of the nuclear enzyme, poly (ADP-ribose) polymerase (PARP), that has been implicated in the pathogenesis of multiple organ failure. Thus, we used an immunohistochemical approach to assess the presence of PAR, an indicator of PARP activation in vivo. There was positive staining for PAR localized in sections of pancreas (Figure 13A), lung (Figure 13B) and gut (Figure 13C) obtained from zymosan-challenged mice. Treatment with GW0742 reduced the degree of positive staining for PAR in the pancreas (Figure 13D), lung (Figure 13E) and gut (Figure 13F). No positive staining for PAR was identified in tissues from Sham-operated mice (data not shown).

Immunohistological staining for the Fas Ligand in the pancreas (Figure 14A), lung (Figure 14B) and gut (Figure 14C) were determined 18 h after zymosan-induced injury. Tissue sections from the sham-operated mice did not stain for the Fas Ligand (data not shown), whereas sections obtained from the zymosan-challenged mice exhibited positive staining for the Fas Ligand, in the pancreas (Figures 14A), lung (Figure 14B) and gut (Figure 14C). Treatment with GW0742 reduced the degree of positive staining for the Fas Ligand in the pancreas (Figures 14D), lung (Figure 14E) and gut (Figure 14F).

To test whether tissue damage was associated with cell death by apoptosis, we assessed TUNEL-like staining in pancreas (Figure 15A) and pulmonary tissue (Figure 15B). Almost no apoptotic cells were detectable in sections of pancreatic and pulmonary tissue in sham-operated mice (data not shown). At 18 h after zymosan-induced injury, sections of pancreas (Figure 15A) and lung (Figure 15B) demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments. In contrast, pancreatic (Figure 15C) and pulmonary (Figure 15D) tissue obtained from zymosan-administered mice treated with GW0742, demonstrated a small number of apoptotic cells or fragments.

The presence of Bax and Bcl-2 in lung homogenates was investigated by Western blot 18 hours after zymosan administration. A basal level of Bax was detected in lung tissues obtained from sham-treated animals (Figure 16A, see densitometric analysis Figure 16A1). Bax levels were substantially increased in the lung tissues from zymosan-administered mice (Figure 16A, see densitometric analysis Figure 16A1). In contrast, GW0742 treatment prevented the zymosan-induced Bax expression (Figure 16A, see densitometric analysis Figure 16A1). A basal level of Bcl-2 was detected in lung tissues obtained from sham-treated animals (Figure 16B, see densitometric analysis Figure 16B1). Bcl-2 levels were substantially reduced in the lung tissues from zymosan-administered mice (Figure 16B, see densitometric analysis Figure 16B1). In contrast, GW0742 treatment significantly attenuated the zymosan-induced reduction of Bcl-2 expression (Figure 16B, see densitometric analysis Figure 16B1).

To determine the immunohistological staining for Bax (Figure 17) and Bcl-2 (Figure 18), samples of pancreas (Figure 17A, 18A respectively) lung (Figure 17B, 18B, respectively) and gut (Figure 17C, 18C respectively) were also collected 18 hours after zymosan administration. Tissues taken from sham-treated mice did not stain for Bax (data not shown), whereas pancreatic (Figure 17A), pulmonary (Figure 17B) and intestinal (Figure 17C) sections obtained from zymosan-treated mice exhibited positive staining for Bax. GW0742 treatment reduced the degree of positive staining for Bax in the pancreas (Figure 17D), lung (Figure 17E) and gut (Figure 17F) of mice subjected to zymosan-induced injury.

In addition, lung sections from sham-treated mice demonstrated positive staining for Bcl-2 (data not shown), whereas in zymosan-administered mice, Bcl-2 staining was significantly reduced (Figure 18). GW0742 treatment significantly attenuated the loss of positive staining for Bcl-2 in pancreatic (Figure 18D), pulmonary (Figure 18E) and intestinal (Figure 18F) samples of mice subjected to zymosan-induced injury.