Role of peroxisome proliferator-activated receptor-α in acute pancreatitis induced by cerulein

Abstract

The peroxisome proliferator-activated receptor-α (PPAR-α) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The aim of the present study was to examine the effects of endogenous PPAR-α ligand on the development of acute pancreatitis caused by cerulein in mice. Intraperitoneal injection of cerulein into PPAR-α wild-type (WT) mice resulted in severe, acute pancreatitis characterized by oedema, neutrophil infiltration and necrosis and by elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as an increase in myeloperoxidase activity) was associated with enhanced expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) in the pancreas of cerulein-treated PPAR-α wild-type (WT) mice in comparison to sham-treated mice. Acute pancreatitis in PPAR-αWT mice was also associated with a significant mortality (20% survival at 5 days after cerulein administration). In contrast, the degree of pancreatic inflammation and tissue injury (histological score), up-regulation/formation of ICAM-1 and P-selectin, infiltration of neutrophils, and the expression of TGF-β and VEGF was markedly enhanced in pancreatic tissue obtained from cerulein-treated PPAR-α knockout (KO) mice. Thus, endogenous PPAR-α ligands reduce the degree of pancreas injury caused by acute pancreatitis induced by cerulein administration.

Introduction

Acute pancreatitis is a common cause of emergency hospital admission, with an annual incidence of 41·9 cases per 100 000 population.¹ Various studies have demonstrated that growth factors are involved in pancreatic development, growth and regeneration.^2–4 In particular, several studies have pointed out that various growth factors, such as fibroblast growth factor-1 (FGF-1),^5,6 fibroblast growth factor-2 (FGF-2),^5,6 insulin-like growth factor-1 (IGF-1),^5–7 hepatocyte growth factor (HGF),^5,6 transforming growth factor-α (TGF-α),^5,6 transforming growth factor-β (TGF-β)^7,8 and epidermal growth factor (EGF),^7,8 are overexpressed in the pancreatic tissues from animals subjected to experimental acute pancreatitis. In addition, clinical studies have also demonstrated a significant increase in the pancreatic expression of growth factors, such as TGF-β,^9–12 connective tissue growth factor (CTGF),¹³ FGF-1 and FGF-2¹¹ in pancreatic samples obtained from patients with acute necrotizing pancreatitis. Other important candidate factors that may modulate pancreatic injury are pro-inflammatory cytokines [such as tumour necrosis factor-α (TNF-α), interleukin (IL)-1 and IL-6]¹² known to be up-regulated early in the course of acute pancreatic necroinflammation. Recently, the availability of the prototypical anti-TNF agents has offered an important potential advance in the therapy for patients with acute pancreatitis. In particular, it has been shown that infliximab, a chimeric monoclonal anti-TNF-α immunoglobulin, significantly reduces the development of acute pancreatitis as well as the associated lung injury.¹³ In addition, recruitment of inflammatory cells from the circulation is an important process to augment the inflammatory response.¹⁴ TNF-α induces the expression of adhesion molecules in the vascular endothelium, and invasion of inflammatory cells into inflamed tissues subsequently occurs. P-selectin, a member of the selectin family of adhesion molecules, and intercellular adhesion molecule-1 (ICAM-1), both of which are expressed at the surface of the vascular endothelium, are involved in this process.¹⁵ Various mediators contribute to the up-regulation of endothelial cell and leucocyte-adhesion molecules in inflammation. Several mediators, including growth factors [e.g. vascular endothelial growth factor (VEGF)], cytokines (e.g. TNF-α) and serine proteases (e.g. thrombin), activate gene transcription in endothelial cells, resulting in changes in the haemostatic balance, increased leucocyte adhesion, loss of barrier function, increased permeability, migration, proliferation and successive angiogenesis. Under normal conditions, the activation signal may be terminated by negative feedback inhibition of downstream transcriptional networks. Such a mechanism has been well established for TNF-α.¹⁶ In contrast, little is known about the major self-regulatory processes involved in VEGF and acute pancreatitis. VEGF is an endothelial cell-specific mitogen and chemotactic agent, which is involved in wound repair, angiogenesis of ischemic tissue, tumour growth, microvascular permeability, haemostasis and endothelial cell survival.^17,18 In a recent study, it has been shown that acute pancreatitis was associated with immunostaining for VEGF in acinar and ductal epithelial cells, cells of pancreatic islets, as well as in tubular complexes, inflammatory infiltration cells and granulation tissue.^19,20

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors.^21,22 The PPAR subfamily comprises three members, PPAR-α, PPAR-β and PPAR-γ.²³ In rats, PPAR-α is most highly expressed in brown adipose tissue, followed by liver, kidney, heart and skeletal muscle.²⁴ Recently, it has been demonstrated that PPAR-α is also expressed in the digestive tract, being localized mainly in the intestinal mucosa, the small intestine and the colon,²⁵ as well as in the pancreas. PPAR-α binds to a diverse set of ligands, namely arachidonic acid metabolites (prostaglandins and leukotrienes) and plasticizers and synthetic fibrate drugs, including clofibrate, fenofibrate and bezafibrate.²⁶

While PPAR-α has been studied less than PPAR-γ, PPAR-α ligands have been shown to regulate inflammatory responses.²⁷ In addition, we and other authors have clearly demonstrated that PPAR-α-deficient mice have abnormally prolonged responses to different inflammatory stimuli.^28–31 However, the role of endogenous PPAR-α ligands in experimental acute pancreatitis have not yet been investigated.

In this study, we investigated the role of endogenous PPAR-α ligands in a model of acute pancreatitis using PPAR-α knockout (KO) mice. In order to characterize the role of endogenous PPAR-α ligands in this model of acute pancreatitis, we determined the following end-points of the inflammatory response: serum amylase and lipase levels; neutrophil infiltration; adhesion molecule expression; TNF-α production; VEGF and TGF-β production; and organ injury and survival.

Materials and methods

Animals

Mice (4–5 weeks old, 20–22 g) with a targeted disruption of the PPAR gene (PPAR-αKO) and littermate wild-type controls (PPAR-αWT) were purchased from Jackson Laboratories (Harlan Nossan, Italy). Mice homozygous for the PparatniJGonz targeted mutation are viable, fertile and appear normal in appearance and behaviour. Exon 8, encoding the ligand-binding domain, was disrupted by the insertion of a 1·14-kb neomycin-resistance gene in the opposite transcriptional direction. After electroporation of the targeting construct into J1 ES cells, the ES cells were injected into C57BL/6 N blastocysts. This stain was created on a B6, 129S4 background and is maintained as a homozygote on a 129S4/SvJae background by brother–sister mating. The study was approved by the University of Messina Review Board for the care of animals. The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986) and the USA (Animal Welfare Assurance No A5594-01, Department of Health and Human Services).

Induction of pancreatitis

Mice were randomly allocated into the following groups.

1. PPAR-αWT + cerulein group. Mice were treated hourly (× 5) with cerulein (50 µg/kg, suspended in saline solution, intraperitoneally) (n = 10).

2. PPAR-αKO + cerulein group. Mice were treated hourly (× 5) with cerulein (50 µg/kg, suspended in saline solution, intraperitoneally) (n = 10).

3. PPAR-αWT + saline group. The sham-treated group in which identical treatments to the cerulein group were performed, except that saline was administered instead of cerulein.

4. PPAR-αKO + saline group. Identical to the PPAR-αWT + saline group, except for the use of PPAR-αKO mice.

Mice were killed by exsanguination 6 hr after the induction of pancreatitis. Blood samples were obtained by direct intracardiac puncture. Pancreas and lungs were removed immediately, frozen in liquid nitrogen, and stored at −80° until assayed. Portions of these organ were also fixed in formaldehyde for histological and immunohistochemical examination. In another set of experiments, mice were randomized to receive treatment regimens identical to those listed above (n = 20 for each group), but were monitored for 5 days in order to monitor their survival rate.

Morphological examination

Paraffin-embedded pancreas samples were sectioned (5 µm) and stained with haematoxylin and eosin. Pancreas sections were examined by an experienced morphologist, who was not aware of the sample identity. Acinar-cell injury/necrosis was quantified by morphometry, as previously described.³² For these studies, 10 randomly chosen microscopic fields (× 125 magnnification) were examined for each tissue sample, and the extent of acinar-cell injury/necrosis was expressed as the percentage of the total acinar tissue. The criteria for injury/necrosis were as follows: the presence of acinar-cell ghosts; or vacuolization and swelling of acinar cells and the destruction of the histoarchitecture of whole or parts of the acini, both of which had to be associated with an inflammatory reaction.

Determination of pancreas oedema

The extent of pancreatic oedema was assayed by measuring the difference between wet and dry pancreas tissue weight. For these latter measurements, a freshly obtained blotted sample of pancreas was weighted on aluminium foil, dried for 12 hr at 95°, and reweighed. The difference between wet and dry tissue weight was calculated and expressed as a percentage of tissue wet weight.

Localization of P-selectin, ICAM-1, VEGF and TGF-β by immunohistochemistry

Six hours after cerulein administration, the pancreas tissues were fixed in 10% (w/v) phosphate-buffered saline (PBS, 0·01 m, pH 7·4)-buffered formaldehyde and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched, for 30 min, with 0·3% (v/v) hydrogen peroxide in 60% (v/v) methanol. The sections were permeabilized with 0·1% (w/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- and avidin-binding sites were blocked by sequential incubation for 15 min with avidin and biotin, respectively (DBA, Milan, Italy). Sections were incubated overnight with anti-P-selectin immunoglobulin (1 : 500 in PBS, v/v), mouse anti-rat immunoglobulin directed at ICAM-1 (CD54) (1 : 500 in PBS, v/v) (DBA), anti-TGF-β (1 : 500 in PBS, v/v) (DBA), or anti-VEGF immunoglobulin (1 : 500 in PBS, v/v) (DBA). Specific labelling was detected with a biotin-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (IgG) and avidin–biotin peroxidase complex (DBA). To verify the binding specificity for ICAM-1, P-selectin, VEGF and TGF-β, some sections were also incubated with primary antibody only (no secondary) or with secondary antibody only (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreactions were positive in all the experiments carried out.

Immunocytochemistry photographs (n = 5) were assessed by densitometry, as previously described.³³ The assay was carried out by using optilab graftek software on a Macintosh personal computer.

Total protein extraction and western blot analysis for P-selectin and TGF-β

Tissue samples obtained from animals 6 hr after cerulein administration were homogenized with an Ultra-turrax T8 homogenizer in a buffer containing 20 mm Tris-HCl, pH 7·4, 150 mm NaCl, 0·1% Triton X-100, 1 mm phenylmethylsulphonyl fluoride (PMSF), 1·5 µg/ml trypsin inhibitor, 3 µg/ml pepstatin, 2 µg/ml leupeptin and 40 µm benzidamin. The homogenates were centrifuged (17 400 g/min, 15 min, 4°), and the supernatant was collected for evaluating the concentration of P-selectin and TGF-β.

Protein concentration was determined using the Bio-Rad (Milan, Italy) protein assay kit. Proteins were mixed with gel-loading buffer [50 mm Tris, 10% (w/v), sodium dodecyl sulphate (SDS), 10% (w/v) glycerol, 10% (v/v) 2-mercaptoethanol and 2 mg/ml bromophenol], boiled for 3 min and centrifuged at 10 300 g/min for a few seconds. Protein concentration was determined, and equivalent amounts (50 µg) of each sample were electrophoresed in a 12% (w/v) discontinuous polyacrylamide minigel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked with 10% nonfat dry milk in Tris-buffered saline (TBS) for 1 hr and incubated with primary antibodies against P-selectin and TGF-β (at a 1 : 100 dilution) overnight at 4°. The membranes were washed three times for 10 min in TBS with 0·1% Tween 20 and incubated with AffiniPure Goat Anti-Rabbit IgG coupled to peroxidase (1 : 2000).

The primary antibodies directed at P-selectin and TGF-β were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The secondary antibody was obtained from Jackson Immuno Research, Laboratories, Inc. (Jackson, PA). The immune complexes were visualized using enhanced chemiluminescence (ECL) (Amersham, Bucks, UK). Subsequently, the relative expression of the proteins was quantified by densitometric scanning of the X-ray films with a GS-700 Imaging Densitometer (Bio-Rad) and a computer program (molecular analyst; IBM, Milan, Italy).

Quantification of ICAM-1 and VEGF protein levels

The pancreatic tissue was thawed, placed in complete lysis buffer (Boehringer Mannheim, Indianapolis, IN) homogenized with a hand-held homogenizer and sonicated for 10 s, as previously described.³⁴ The homogenate was centrifuged at 10 000 g for 5 min, and the supernatant and sediment fraction were separated. Quantification of ICAM-1 and VEGF were normalized to total protein in the sample. Total protein determination was performed using a modified Bradford assay with serial dilutions of bovine serum albumin.

The assay was carried out by using a colorimetric, commercial kit (R & D Systems, Milan, Italy) according to the manufacturer's instructions. The reaction was stopped, and the absorption was measured in an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm. VEGF and ICAM-1 determinations were performed in duplicate serial dilutions.

Myeloperoxidase activity

Myeloperoxidase activity, an index of polymorphonuclear leucocyte (PMN) accumulation, was determined as previously described.³⁵ Pancreas tissues, collected at a specified time point, were homogenized in a solution containing 0·5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mm potassium phosphate buffer (pH 7) and centrifuged (30 min, 20 000 g, 4°). An aliquot of the supernatant was then 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 a spectrophotometer set at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol of peroxide per min at 37° and was expressed in units per gram weight of wet tissue.

Measurement of TNF-α

Plasma and pancreas tissues were collected 6 hr after cerulein administration, and plasma and tissue levels of TNF-α were evaluated. Pancreas tissues were homogenized, as previously described,³⁶ in PBS containing 2 mmol/l of PMSF (Sigma Chemical Co., Milan, Italy). The assay was carried out by using a colorimetric, commercial kit (R & D Systems) according to the manufacturer's instructions. All TNF-α determinations were performed in duplicate serial dilutions.

Materials

Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company (Milan, Italy). Primary monoclonal ICAM-1 (CD54) for immunohistochemistry was purchased from Pharmingen (Milan, Italy). Reagents, and secondary and non-specific IgG for immunohistochemical analysis, were from Vector Laboratories Inc. (DBA, Milan, Italy). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0·9% NaCl; Baxter Healthcare Ltd, Thetford, Norfolk, UK).

Data analysis

All values in the figures and text are expressed as mean ± standard error (SEM) of the mean of n observations. In 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 performed on different experimental days. The results were analysed by one-way analysis of variance (anova) followed by a Bonferroni posthoc test for multiple comparisons. A P-value of less than 0·05 was considered significant.

Results

Effects of endogenous PPAR-α ligand on the degree of acute pancreatitis

Cerulein-induced pancreatitis in PPAR-αWT mice was associated with significant increases in the serum levels of lipase and amylase (Fig. 1). The increase in lipase and amylase was markedly enhanced in cerulein-treated PPAR-αKO mice (Fig. 1). In sham-saline mice the histological features of the pancreas were typical of a normal architecture (Fig. 2c). As described previously,³⁷ mice treated with intraperitoneal injections of the secretagogue cerulein develop acute necrotizing pancreatitis. Histological examination (6 hr after the injection of cerulein) of pancreas sections from PPAR-αWT mice revealed tissue damage characterized by inflammatory cell infiltrates and acinar cell necrosis (Fig. 2a,a1; Table 1).

Figure 1.

Amylase (a) and lipase (b) serum levels (U/l). The absence of the peroxisome proliferator-activated receptor-α (PPAR-α) gene significantly enhanced the increase of amylase and lipase induced by cerulein. Data represent the mean ± standard error of the mean (SEM) of 10 animals for each group. *P < 0·01 versus sham-treated mice. †P < 0·01 versus wild-type (WT) mice. KO, knockout.

Figure 2.

Morphological changes of pancreas 6 hr after the administration of cerulein. The pancreas section from cerulein-treated mice showed interstitial oedema (a) and infiltration of the tissue with inflammatory cells (a1). The absence of the peroxisome proliferator-activated receptor-α (PPAR-α) gene significantly increased the extent and severity of the histological signs of pancreas injury (b,b1). No histological alterations were observed in the pancreas tissues from sham-treated mice (c). The figure is representative of at least three experiments performed on different experimental days.

Table 1.

Histological scoring of the acute pancreatitis lesion

	Oedema	Inflammation	Necrosis
CER + WT	1·6 ± 0·13	1·9 ± 0·11	1·97 ± 0·1
CER + KO	2·9 ± 0·12*	2·35 ± 0·1*	2·35 ± 0·09*

Oedema, inflammation and necrosis were evaluated 6 hr after injection with cerulein (CER). The absence of the proliferator-activated receptor-α (PPAR-a) gene significantly increased the extent and severity of the histological score of pancreas injury.

^*P < 0·01 versus cerulein + wild-type (WT) mice. KO, knockout.

Furthermore, the injection of cerulein elicited an inflammatory response, characterized by oedema formation in the pancreas tissues (Fig. 3). The absence of the PPAR-α gene significantly increased the extent and severity of the histological signs of pancreas injury (Fig. 2b,b1; Table 1) as well as the accumulation of water in the pancreas (Fig. 3).

Figure 3.

Effects of endogenous peroxisome proliferator-activated receptor-α (PPAR-α) ligand on oedema formation in the pancreas 6 hr after cerulein-induced acute pancreatitis. Data represent the mean ± standard error of the mean (SEM) of 10 animals for each group. *P < 0·01 versus sham-treated mice. †P < 0·01 versus wild-type (WT) mice. KO, knockout.

Endogenous PPAR-α ligand modulates production of TNF-α after cerulein administration

To test whether endogenous PPAR-α ligand may modulate the inflammatory process through the regulation of cytokine secretion, we analysed the pancreas levels of the pro-inflammatory cytokine TNF-α in PPAR-αKO and PPAR-αWT mice. A substantial increase of TNF-α formation was found in pancreas samples collected from PPAR-αWT mice 6 hr after cerulein administration (Fig. 4). Pancreas levels of TNF-α were significantly higher in PPAR-αKO mice compared with those of PPAR-αWT mice (Fig. 4).

Figure 4.

Effects of endogenous peroxisome proliferator-activated receptor-α (PPAR-α) ligand on pancreas tumour necrosis factor-α (TNF-α) production. PPAR-α wild-type (WT) mice show a significant production of TNF-α 6 hr after the administration of cerulein. The genetic absence of the PPAR-α receptor resulted in a significant enhancement in the production of pro-inflammatory cytokine. Data represent the mean ± standard error of the mean (SEM) of 10 mice for each group. *P < 0·01 versus sham-treated mice. †P < 0·01 versus wild-type (WT) mice. KO, knockout.

Effects of endogenous PPAR-α ligand on P-selectin and ICAM-1 expression and neutrophil infiltration

A hallmark of acute pancreatitis is the accumulation of neutrophils in the pancreas, which augments the tissue damage. Therefore, we evaluated the extent of expression of P-selectin and ICAM-1, adhesion molecules that play a pivotal role in the rolling and firm attachment of neutrophils to the endothelium. No positive staining for P-selectin was found in pancreas tissue sections from saline-treated mice (data not shown). Six hours after cerulein injection, positive staining for P-selectin was observed along the vessels (Figs 5a and 6) in the pancreas collected from PPAR-αWT mice. The immunostainings for P-selectin (Figs 5b and 6) were markedly enhanced in pancreas tissues from cerulein-treated PPAR-αKO mice. A significant increase in P-selectin expression 6 hr after cerulein injection, assayed by western blot analysis, was also detected in pancreas obtained from PPAR-αWT mice subjected to cerulein-induced acute pancreatitis (Fig. 5c,c1). The absence of the PPAR-α receptor enhanced the P-selectin expression in the pancreas (Fig. 5c,c1). Staining of pancreas tissue sections, obtained from sham-treated mice, with anti-ICAM-1 immunoglobulin showed a specific staining along the bronchial epithelium, demonstrating that ICAM-1 is constitutively expressed (data not shown). Six hours after cerulein injection, the staining intensity for ICAM-1 substantially increased along the vessels in the pancreas collected from PPAR-αWT mice (Figs 6 and 7a). The immunostainings for ICAM-1 (Figs 6 and 7b) were markedly enhanced in pancreas tissues from cerulein-treated PPAR-αKO mice. ICAM-1 levels in the pancreas from sham-treated PPAR-αWT and PPAR-αKO were detectable only at very low levels. Injection of cerulein markedly enhanced the pancreatic content of ICAM-1 (Fig. 7c). The absence of PPAR-α significantly enhanced the pancreatic content of ICAM-1 (Fig. 7c).

Figure 5.

Immunohistochemical localization of P-selectin in the pancreas 6 hr after the administration of cerulein. The section obtained from cerulein-treated peroxisome proliferator-activated receptor-α (PPAR-α) wild-type (WT) mice showed intense positive staining for P-selectin (a) in the pancreas. The degree of pancreas staining for P-selectin (b) was markedly enhanced in pancreas tissue sections obtained from cerulein-treated PPAR-α knockout (KO) mice. A representative blot of P-selectin (c,c1). The genetic absence of the PPAR-α receptor significantly enhanced the expression of P-selectin in the pancreas (c,c1). (c1) The intensity of retarded bands (measured by phosphoimager) in all the experimental groups. Immunoblotting in panel c is representative of one pancreas tissue out of five or six analysed. The results in panel c1 represent the mean ± standard error of the mean (SEM) from five or six pancreas tissues. *P < 0·01 versus cerulein-treated PPAR-α WT mice.

Figure 6.

Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n = 5) for P-selectin (P-SEL), intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) from pancreas tissues. The assay was carried out by using optilab graftek software on a Macintosh personal computer (CPU G3-266). ND, not detected. Data are expressed as the percentage of the total tissue area. *P < 0·01 versus sham-treated mice. †P < 0·01 versus wild-type (WT) mice. KO, knockout.

Figure 7.

Immunohistochemical localization of intercellular adhesion molecule-1 (ICAM-1) in the pancreas 6 hr after the administration of cerulein. Sections obtained from cerulein-treated peroxisome proliferator-activated receptor-α (PPAR-α) wild-type (WT) mice showed intense positive staining for ICAM-1 (a) in the pancreas. The degree of pancreas staining for ICAM-1 (b) was markedly enhanced in pancreas tissue sections obtained from cerulein-treated PPAR-α knockout (KO) mice. Similarly, a significant increase of ICAM-1, assessed by enzyme-linked immunosorbent assay (ELISA), was observed in pancreas tissues from cerulein-treated PPAR-αWT mice in comparison with sham-treated mice (c). The absence of the PPAR-α receptor significantly enhanced the pancreatic content of ICAM-1 (c). Data represent the mean ± standard error of the mean (SEM) of 10 mice for each group. *P < 0·01 versus sham-treated mice. †P < 0·01 versus WT mice.

The expression of adhesion molecules in the pancreas appeared to be correlated with the influx of leucocytes into the pancreas tissue. Therefore, we investigated the effect of endogenous PPAR-α on the infiltration of neutrophils by measurement of the activity of myeloperoxidase. Myeloperoxidase activity was significantly elevated 6 hr after cerulein administration in PPAR-αWT mice (Fig. 8). Neutrophil infiltration in pancreas tissue was significantly enhanced in PPAR-α-deficient mice compared with those of PPAR-αWT animals (Fig. 8).

Figure 8.

Effect of endogenous peroxisome proliferator-activated receptor-α (PPAR-α) ligand on neutrophil infiltration. Myeloperoxidase (MPO) activity was significantly increased in the pancreas from cerulein-treated PPAR-α wild-type (WT) mice in comparison to that of sham-treated mice. The genetic absence of the PPAR-α receptor significantly enhanced the cerulein-induced increase of MPO activity in the pancreas. Data represent the mean ± standard error of the mean (SEM) of 10 mice for each group. *P < 0·01 versus sham-treated mice. †P < 0·01 versus WT mice. KO, knockout.

Effects of endogenous PPAR-α ligand on TGF-β and VEGF expression

To assess whether endogenous PPAR-α ligands modulate the leucocyte adhesion and infiltration through a reduction in TGF-β and VEGF levels, we evaluated the expression of TGF-β and VEGF in the pancreas. No positive staining for TGF-β was found in pancreas section from saline-treated mice (data not shown). Six hours after cerulein injection, positive staining for TGF-β was found to be localized mainly in the acinar and ductal cells as well as in the vessel walls of the inflamed pancreas collected from PPAR-αWT mice (Figs 6 and 9a). The immunostainings for TGF-β (Figs 6 and 9b) were markedly enhanced in pancreas tissues from cerulein-treated PPAR-αKO mice. A significant increase in TGF-β expression 6 hr after cerulein injection, assayed by western blot analysis, was also detected in pancreas obtained from PPAR-αWT mice subjected to cerulein-induced acute pancreatitis (Fig. 9c,c1). The absence of the PPAR-α receptor enhanced the P-selectin expression in the pancreas (Fig. 9c,c1). No positive staining for VEGF was observed in the pancreas tissue sections obtained from sham-treated mice (data not shown). Six hours after injection with cerulein, the staining intensity for VEGF substantially increased in the acinar and ductal cells, as well as in the vessel wall of the inflamed pancreas (Figs 6 and 10a). The immunostainings for VEGF (Figs 6 and 10b) were markedly enhanced in pancreas tissues from cerulein-treated PPAR-αKO mice. VEGF was detectable in the pancreas from sham-treated PPAR-αWT and PPAR-αKO. However, injection with cerulein markedly enhanced the pancreatic content of VEGF (Fig. 10c). The absence of PPAR-α significantly enhanced the pancreatic content of VEGF (Fig. 10c).

Figure 9.

Immunohistochemical localization of transforming growth factor-β (TGF-β) in the pancreas 6 hr after the administration of cerulein. Sections obtained from cerulein-treated peroxisome proliferator-activated receptor-α (PPAR-α) wild-type (WT) mice showed intense positive staining for TGF-β (a) in the pancreas. The degree of pancreas staining for TGF-β (b) was markedly enhanced in pancreas tissue sections obtained from cerulein-treated PPAR-α knockout (KO) mice. A representative blot of TGF-β (c,c1). The genetic absence of the PPAR-α receptor significantly enhanced the TGF-β expression in the pancreas (c,c1). (c1) The intensity of retarded bands (measured by phosphoimager) in all the experimental groups. Immunoblotting in panel c is representative of one pancreas tissue out of five or six analysed. The results in panel c1 are expressed as the mean ± standard error of the mean (SEM) from five or six pancreas tissues. *P < 0·01 versus cerulein-treated PPAR-αWT mice. KO, knockout.

Figure 10.

Immunohistochemical localization of vascular endothelial growth factor (VEGF) in the pancreas 6 hr after the administration of cerulein. Sections obtained from cerulein-treated peroxisome proliferator-activated receptor-α (PPAR-α) wild-type (WT) mice showed intense positive staining for VEGF (a) in the pancreas. The degree of pancreas staining for VEGF (b) was markedly enhanced in pancreas tissue sections obtained from cerulein-treated PPAR-α knockout (KO) mice. VEGF levels in the pancreas from sham-treated PPAR-α wild-type (WT) and PPAR-αKO mice were detectable only at very low levels. Injection of cerulein markedly enhanced the pancreatic content of VEGF (c). The absence of PPAR-α significantly enhanced the pancreatic content of VEGF (c). Data represent the mean ± standard error of the mean (SEM) of 10 mice for each group. *P < 0·01 versus sham-treated mice. †P < 0·01 versus WT mice.

Effects of endogenous PPAR-α ligand on survival rate

The survival of animals was monitored for 5 days. Cerulein-treated PPAR-αWT mice developed severe acute pancreatitis and 70% of these animals died within 5 days after cerulein administration (Fig. 11). The absence of a functional PPAR-α gene in PPAR-αKO mice resulted in a significant augmentation of the mortality rate (Fig. 11).

Figure 11.

Effects of endogenous proliferator-activated receptor-α (PPAR-α) ligand on cerulein-induced mortality. The absence of the PPAR-α gene significantly enhanced the cerulein-induced mortality. Data are expressed as the mean ± standard error of the mean (SEM) of 20 mice for each group. P < 0·01 versus cerulein-treated PPAR-α wild-type (WT) mice. KO, knockout.

Discussion

We demonstrate, in this study, that the absence of the PPAR-α gene significantly increased cerulein-induced pancreatitis, pancreas injury, the production of pro-inflammatory cytokines, TGF-β and VEGF expression, neutrophil infiltration, the increased expression of ICAM-1 and P-selectin, and the degree of mortality. All of these findings support the view that endogenous PPAR-α ligand exerts potent anti-inflammatory effects. What, then, is the mechanism by which PPAR-α ligand inhibits the pancreas injury caused by acute pancreatitis?

Recently, several studies have reported that PPARα activators suppress IL-1-induced C-reactive protein (CRP) and IL-6-induced fibrinogen expression, the major acute-phase response (APR) proteins in humans,³⁸ whose plasma concentrations are elevated not only in acute but also in chronic inflammatory states. This anti-inflammatory action of PPAR-α is not restricted to these genes, but also applies more generally to other APR genes, such as serum amyloid A (SAA) and fibrinogen-a and -b.³⁹ PPAR-α activation leads to a reduction in the formation of nuclear C/EBPbBp50/nuclear factor-kappa B (NF-κB) complexes, and thereby reduces the activation of the CRP promoter. Moreover, PPAR-α increases IkB-α expression, thus preventing nuclear p50/p65 NF-κB translocation and arresting their nuclear transcriptional activity. Chronic treatment with fibrates decreases hepatic C/EBPb and p50-NF-κB protein expression in mice in a PPARα-dependent manner.³⁹ This latter effect probably contributes to the generalized anti-inflammatory effects of fibrates on the expression of a wide range of APR genes containing response elements for these transcription factors in their promoters. NF-κB activation induces the transcription of many pro-inflammatory genes, including TNF-α, IL-1β and ICAM-1, to name but a few.⁴⁰

We report, in the present study, that endogenous PPAR-α ligand reduces (among other effects) the biosynthesis and/or the effects of the pro-inflammatory cytokine, TNF-α. There is good evidence that TNF-α is clearly involved in the pathogenesis of acute pancreatitis. Direct evidence that TNF-α plays a role in the pathogenesis of acute pancreatitis has been obtained in animal models in which blocking of the action of this cytokine has been shown to delay the onset of acute pancreatitis.¹³

In addition, recently it has also been demonstrated that TGF-β plays a critical role in the development of pancreatitis, which was detected in human specimens of acute pancreatitis and experimental pancreatitis animal models.^41,42 In addition, the increase of TGF-β associated with the expression of collagen and fibronectin has been shown during tissue regeneration after acute pancreatitis.⁴¹ Moreover, recently it has been demonstrated that the conditional loss of TGF-β signalling selectively in the pancreas led to amelioration of pancreatic fibrosis in mice.²⁰ We confirm, in the present study, that the model of acute pancreatitis used here leads to a substantial increase in the levels of TNF-α and TGF-β in the pancreas. Interestingly, the levels of these pro-inflammatory cytokines are significantly higher in the absence of a functional PPAR-α gene. These findings therefore suggest that endogenous PPAR-α ligand reduced the activation and the subsequent expression of pro-inflammatory genes. Neutrophils play a crucial role in the development and full manifestation of acute pancreatitis.⁴³ Neutrophil infiltration into inflamed tissue plays a crucial role in the destruction of foreign antigens and in the breakdown and remodeling of injured tissue. The adhesion molecules ICAM-1 and P-selectin play an important role in the recruitment of neutrophils into the pancreas during acute pancreatitis after ischemia and reperfusion.⁴⁴ In this study, as previously published,^43,44 we demonstrate that experimental acute pancreatitis leads to an increased expression of ICAM-1 and P-selectin, which also results in a significant neutrophil infiltration. Thus, the enhanced expression of ICAM-1 and P-selectin, as well as the increased neutrophil infiltration observed in the absence of endogenous PPAR-α ligand, suggests that endogenous PPAR-α ligand reduced neutrophil infiltration, modulating the expression and the up-regulation of adhesion molecules.

In addition, recent studies have indicated that serum concentrations of VEGF increased from the normal level to threefold and 1·5-fold, respectively, during the first week of hospitalization in patients with acute pancreatitis.⁴⁵ However, in this study, the authors did not observe any relationship between serum VEGF levels and disease severity.⁴⁵ In the present study, we demonstrated that experimental acute pancreatitis leads to an increased expression of VEGF, which also results in a significant pancreas oedema. Thus, the enhanced expression of VEGF, as well as the increased pancreas oedema observed in the absence of endogenous PPAR-α ligand, suggests that endogenous PPAR-α ligand reduced the vascular permeability and oedema formation also modulating the expression of VEGF. In conclusion, this study provides evidence that endogenous PPAR-α ligand, like exogenous ligand, caused a substantial reduction of pancreas injury associated with experimental acute pancreatitis. Thus, we demonstrate here that the mechanisms underlying the protective effects of endogenous PPAR-α ligand are dependent on the activation of PPAR-α. The activation of PPAR-α by endogenous ligands in turn, results in a reduction of the formation of the pro-inflammatory cytokines, the expression of the adhesion molecule, ICAM-1, expression of VEGF and neutrophil infiltration.

Abbreviations

APR

acute-phase response

CRP

C-reactive protein

CTGF

connective tissue growth factor

EGF

epidermal growth factor

FGF-1

fibroblast growth factor-1

FGF-2

fibroblast growth factor-2

HGF

hepatocyte growth factor

IGF-1

insulin-like growth factor-1

ICAM-1

intercellular adhesion molecule-1

PBS

phosphate-buffered saline

IL

interleukin

NF-κB

nuclear factor kappa B

PMN

polymorphonuclear leucocyte

PMSF

phenylmethylsulphonyl fluoride

PPARs

peroxisome proliferator-activated receptors

PPAR-α

peroxisome proliferator-activated receptor-α

SAA

serum amyloid A

SDS

sodium dodecyl sulphate

TBS

Tris-buffered saline

TGF-β

transforming growth factor-β

TNF-α

tumour necrosis factor-α

VEGF

vascular endothelial growth factor