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Originally published online as doi:10.1189/jlb.0605341 on February 24, 2006

Published online before print February 24, 2006
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(Journal of Leukocyte Biology. 2006;79:999-1010.)
© 2006 by Society for Leukocyte Biology

The role of the peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) in the regulation of acute inflammation

Salvatore Cuzzocrea*,§,1, Emanuela Mazzon*,§, Rosanna Di Paola*, Angelo Peli{dagger}, Andrea Bonato{dagger}, Domenico Britti{ddagger}, Tiziana Genovese*, Carmelo Muià*, Concetta Crisafulli* and Achille P. Caputi*

* Dipartment Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina Torre Biologica, Policlinico Universitario, Italy;
{dagger} Clinical Veterinary Department Alma Mater Studiorum, University of Bologna, Italy; and
{ddagger} Department of Veterinary Clinical Science, University of Teramo, Italy; and
§ Centro per lo Studio edil Trattamento dei Neurolesi Lungodegenti, Facoltà di Medicina e Chirurgia, University of Messina, Italy

1Correspondence: Institute of Pharmacology, School of Medicine, University of Messina, via C. Valeria, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy. E-mail: salvator{at}unime.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) 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 evaluate the role of the PPAR-{alpha} receptor on the development of acute inflammation. To address this question, we used two animal models of acute inflammation (carrageenan-induced paw edema and carrageenan-induced pleurisy). We report here that when compared with PPAR-{alpha} wild-type mice, PPAR-{alpha} knockout mice (PPAR-{alpha}KO) mice experienced a higher rate of the extent and severity when subjected to carrageenan injection in the paw edema model or to carrageenan administration in the pleurisy model. In particular, the absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant augmentation of various inflammatory parameters (e.g., enhancement of paw edema, pleural exudate formation, mononuclear cell infiltration, and histological injury) in vivo. Furthermore, the absence of a functional PPAR-{alpha} gene enhanced the staining (immunohistochemistry) for FAS ligand in the paw and in the lung and the expression of tumor necrosis factor {alpha} and interleukin-1ß in the lungs of carrageenan-treated mice. In conclusion, the increased inflammatory response observed in PPAR-{alpha}KO mice strongly suggests that a PPAR-{alpha} pathway modulates the degree of acute inflammation in the mice.

Key Words: carrageenan-induced paw edema • carrageenan-induced pleurisy • cytokines • neutrophil infiltration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inflammatory process is invariably characterized by a production of prostaglandins, leukotrienes (LTs), histamine, bradykinin, and platelet-activating factor and by a release of chemicals from tissues and migrating cells [1 ]. Carrageenan-induced local inflammation is commonly used to evaluate nonsteroidal, anti-inflammatory drugs (NSAID). Therefore, carrageenan-induced local inflammation (paw edema or pleurisy) is a useful model to asses the contribution of mediators involved in vascular changes associated with acute inflammation.

In particular, the initial phase of acute inflammation (0–1 h), which is not inhibited by NSAID, such as indomethacin or aspirin, has been attributed to the release of histamine, 5-hydroxytryptamine, and bradykinin, followed by a late phase (1–6 h) mainly sustained by prostaglandin release and more recently, has been attributed to the induction of inducible cyclooxygenase 2 (COX-2) in the tissue [2 ]. It appears that the onset of the carrageenan acute inflammation has been linked to neutrophil infiltration and the production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide, and hydroxyl radical, as well as to the release of other neutrophil-derived mediators [3 ].

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors, which are related to retinoid, steroid, and thyroid hormone receptors [4 ]. The PPAR subfamily is comprised of three members: PPAR-{alpha}, PPAR-ß, and PPAR-{gamma} [5 ]. The name PPAR is derived from the fact that activation by xenobiotics of PPAR-{alpha} results in peroxisome proliferation in rodent hepatocytes.

In rats, PPAR-{alpha} is most highly expressed in brown adipose tissue, followed by liver, kidney, heart, and skeletal muscle [6 ]. PPAR-{alpha} binds to a diverse set of ligands, namely, arachidonic acid metabolites (prostaglandins and LTs) and plasticizers and synthetic fibrate drugs including clofibrate, fenofibrate, and bezafibrate [7 ]. Although PPAR-{alpha} has been less studied than PPAR-{gamma}, PPAR-{alpha} ligands have also been shown to regulate inflammatory responses [8 ]. Recent evidence had clearly pointed out that PPAR-{alpha} and PPAR-{gamma} are expressed on T cells and that their ligands can inhibit interleukin (IL)-2 production and T cell proliferation [9 , 10 ]. In addition, we and other authors have clearly demonstrated that PPAR-{alpha}-deficient mice have abnormally prolonged responses to different inflammatory stimuli [11 12 13 14 ]. Given that no single high-affinity, natural ligand has been identified for PPAR-{alpha}, it has been proposed that a physiological role of the receptor may be to sense the total flux of dietary fatty acids in key tissues. Furthermore, fibrates are synthetic ligands for PPAR-{alpha} [15 ], which mediates the lipid-lowering activity of these drugs [16 ]. Recently, it has been demonstrated that fibrates have anti-inflammatory properties in vitro [17 , 18 ] as well as in vivo [8 , 19 ]. In particular, it has been demonstrated that the PPAR-{alpha} ligand can inhibit the expression of different proinflammatory genes, e.g., IL-6, vascular cell adhesion molecule (VCAM), and COX-2 in response to cytokine activation [19 ]. Moreover, it has been demonstrated that the anti-inflammatory effect of PPAR-{alpha} ligands is also dependent on the inhibition of functional nuclear factor (NF)-{kappa}B activation, in part, by increasing the expression of inhibitor of {kappa}B{alpha} (I{kappa}B{alpha}) [20 , 21 ]. However, it is important to point out that these drugs may have multiple effects and may not work via PPAR-{alpha}-dependent pathways [22 , 23 ]. However, Wy14,643, like GW7647, has been identified, which shows excellent selectivity for murine and human PPAR-{alpha} [24 ]. In addition, various studies have also showed that Wy14,643 exerts a potent, anti-inflammatory effect in different models of inflammation [25 ]. On the contrary, the role of the PPAR-{alpha} receptor in conditions associated with experimental acute inflammation has, however, not yet been investigated. The present study was designed to gain a better understanding of the possible influence of PPAR-{alpha} in rodent models of paw edema and lung injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Mice (4–5 weeks old, 20–22 g) with a targeted disruption of the PPAR-{alpha} gene knockout mice (PPAR-{alpha}KO) and littermate wild-type controls (WT) were purchased from Jackson Laboratories (Harlan Nossan, Italy). Mice homozygous for the PparatniJGonz-targeted mutation mice are viable and fertile and appear normal in appearance and behavior. Exon eight, 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/6N blastocysts. This stain was created by Jackson Laboratories on a B6,129S4 background and is maintained as a homozygote on a 129S4/SvJae background by brother-sister matings. The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations for the protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the European Economic Community regulations (O.J. of E.C. L 358/1 12/18/1986).

Experimental groups for carrageenin-induced paw edema
Mice were allocated randomly into the following groups: (i) WT group. WT were subjected to subplantar injection into the right hind paw of 0.1 ml sterile saline containing 1% {lambda}-carrageenan (n=10); (ii) PPAR-{alpha}KO group. PPAR-{alpha}KO were subjected to subplantar injection into the right hind paw of 0.1 ml sterile saline containing 1% {lambda}-carrageenan (n=10); (iii) WT sham group. WT mice were subjected to subplantar injection with the same volume of sterile saline instead of the 1% {lambda}-carrageenan (n=10); (iv) PPAR-{alpha}KO were subjected to subplantar injection with the same volume of sterile saline instead of the 1% {lambda}-carrageenan (n=10).

Carrageenan-induced paw edema
Paw edema was induced as described previously [27 ] by subplantar injection into the mice right hind paw of 0.1 ml sterile saline containing 1% {lambda}-carrageenan. The volume of the paw was measured by a mouse plethysmometer (Basile, Italy) as described previously [27 ] immediately before the subplantar injection and at 1-h intervals up to 5 h. The increase in paw volume was evaluated as the difference between the paw volume at each time-point and the basal paw volume (Time 0). The increase in paw volume was taken as edema volume.

Experimental groups for carrageenin-induced pleurisy
Mice were allocated randomly into the following groups: (i) WT carrageenan group. WT were subjected to carrageenan-induced pleurisy (n=10); (ii) PPAR-{alpha}KO carrageenan group. PPAR-{alpha}KO will be subjected to carrageenan-induced pleurisy (n=10); (iii) WT sham group. WT mice were subjected to the surgical procedures as the above groups except instead of carrageenan, 100 µl saline solution was administered to the mice (n=10); (iv) PPAR-{alpha}KO sham group was subjected to the surgical procedures as the above groups except that instead of carrageenan, 100 µl saline solution was administered to the mice (n=10).

Carrageenan-induced pleurisy
Carrageenan-induced pleurisy was induced as described previously [28 ]. Mice were anesthetized with isoflurane and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.2 ml) or saline containing 1% (w/v) {lambda}-carrageenan (0.2 ml) was injected into the pleural cavity. The skin incision was closed with a suture, and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO2. The chest was opened carefully, and the pleural cavity was rinsed with 2 ml saline solution containing heparin (5 U/ml) and indomethacin (10 µg/ml). The exudate and washing solution were removed by aspiration, and the total volume was measured. Any exudate, which was contaminated with blood, was discarded. The amount of exudate was calculated by subtracting the volume injected (2 ml) from the total volume recovered. The leukocytes in the exudate were suspended in phosphate-buffered saline (PBS; 0.01 M, pH 7.4) and counted with an optical microscope in a Burker’s chamber after vital trypan blue staining.

Histological examination
Paw and lung biopsies were taken 4 h after injection of carrageenan. Tissue biopsies were fixed for 1 week in 10% (w/v) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Sections were then deparaffinized with xylene and stained with trichrome stain (paw sections) or with hematoxylin and eosin (H&E; lung sections). All sections were studied using light microscopy (Dialux 22 Leitz).

Measurement of cytokines
Tumor necrosis factor {alpha} (TNF-{alpha}) and IL-1ß production was evaluated in the pleural exudate at 4 h after the induction of pleurisy by carrageenan injection as described previously [25 ]. The assay was carried out using a colorimetric, commercial enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem-Novabiochem Corp., Milan, Italy) with a lower detection limit of 10 pg/ml.

Immunohistochemical localization of TNF-{alpha}, IL-1, and Fas ligand (FasL)
At the end of the experiment, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde, 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 permeablized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific 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 biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with rabbit anti-IL-1ß antibody (1:500 in PBS, w/v, DBA), with anti-TNF-{alpha} antibody (1:500 in PBS, w/v, Santa Cruz, DBA), or with anti-FasL antibody (1:500 in PBS, v/v, Santa Cruz, DBA). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit immunoglobulin G and avidin-biotin peroxidase complex (DBA). The counter-stain was developed with diaminobenzidine (DAB; brown color) and nuclear fast red (red background). To verify the binding specificity for IL-1ß, TNF-{alpha}, or FasL, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out.

Myeloperoxidase (MPO) activity
MPO activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as described previously [29 ]. At the specified time following injection of carrageenan, paw and lung tissues were obtained and weighed, and each piece was homogenized in a solution containing 0.5% (w/v) hexadecyltrimethyl-ammonium bromide, dissolved in 10 mM potassium phosphate buffer (pH 7), and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM hydrogen peroxide. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol peroxide/min at 37°C and was expressed in units per g wet tissue.

Quantitative real-time polymerase chain reaction (PCR)
Total cellular RNA was isolated from 75 mg homogenized lung tissue using the Trireagent kit (Sigma®, Germany), according to the suggested protocol from the manufacturer’s instructions as described previously [29 , 30 ]. Tissue lysates were kept at –80°C until use. First-strand cDNA synthesis was obtained after a DNase treatment. Retrotranscription was carried out in a total volume of 20 µl containing reverse transcriptase (RT)-PCR buffer 1x, MgCl2 solution (2.5 mM), dithiothreitol solution (10 mM), deoxy-unspecified nucleoside 5'-triphosphate (dNTP) solution (250 µM for each dNTP), 1.25 µM Oligo(dT), RNase inhibitor (10 U), and multiscribe RT (15 U) and stored at –80°C until use [30 ]. Primer pairs for each cytokine were designed using the software Oligo (Molecular Biology Insights, Cascade, CO; ref. [30] and Table 1 ). The PCR reactions were performed using a hot-start Taq polymerase with the following cycle conditions: a denaturation step for 15 min at 95°C and 40 cycles of 30 s denaturation at 94°C, 30 s annealing (TNF-{alpha} 61°C, IL-1ß 61°C, GADPH 56°C), 15 s at 72°C, and 7 min of final extension at 72°C. The real-time PCR assay was developed and evaluated on the Rotor-Gene 3000 system (Corbett Research, Australia). The results of the quantitative real-time PCR of the TNF-{alpha} and IL-1ß mRNA expression are expressed as a ratio between the number of copies of the target cytokine and the number of copies of the housekeeper GAPDH to have an absolute ratio [30 ]


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  		Table 1. Summary of the Real-Time PCR Primer Sequences

 
Statistical evaluation
All values in the figures and text are expressed as mean ± SEM from 10 mice for each group observation. 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 section collected from all the 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of the functional PPAR-{alpha} gene on the course of the carrageenan-induced paw edema
Using a well-established model of acute inflammatory response (carrageenan-induced paw inflammation [31 ]), the putative role of the PPAR-{alpha} receptor ligand in acute inflammation was investigated. No paw edema formation was observed in the sham WT mice and in sham PPAR-{alpha}KO mice (data not shown). Subplantar injection of carrageenan in WT mice leads to a time-dependent development of inflammation, which peaks within 4–5 h (Fig. 1a ). The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice leads to significantly enhanced paw edema formation at all time-points (Fig. 1a) . Therefore, paw tissues were examined for the measurement of MPO activity, the latter being indicative of neutrophil infiltration. As shown in Figure 1b , MPO activity levels were increased significantly (P<0.01) in the paw of WT mice at 4 h after carrageenan injection when compared with sham WT mice and sham PPAR-{alpha}KO mice (Fig. 1b) . Absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant augmentation of MPO activity (Fig. 1b) .


Figure 1
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  		Figure 1. Effects of a functional PPAR-{alpha} gene on paw edema development (a) and neutrophil infiltration (b) induced by carrageenan subplantar injection in the mice. Paw edema was induced by subplantar injection into the right hind paw of 0.1 ml sterile saline containing 1% {lambda}-carageenan. The paw volume was measured before the subplantar injection and at 1-h intervals up to 4 h. The edema volume is the difference in the paw volume at each time-point and the basal paw volume. At 4 h, paw tissue was collected, weighed, and analyzed for MPO activity. Subplantar injection of carrageenan (CAR) in WT mice leads to a time-dependent development of inflammation, which peaks within 4–5 h (a). MPO activity levels were increased significantly in the paw of WT mice at 4 h after carrageenan (b). The absence of a functional PPAR-{alpha} gene resulted in a significant augmentation of the carrageenan-induced paw edema development at all time-points (a). Moreover, MPO activity was enhanced significantly in carrageenan-treated PPAR-{alpha}KO mice (b). #, P < 0.01, versus carrageenan-WT group at the indicated time-points; *, P < 0.01, versus sham; o, P < 0.01, versus carrageenan- WT group.

 
No histological alterations were observed in the paw tissues collected from sham WT mice (Fig. 2a ) and from sham PPAR-{alpha}KO mice (Fig. 2b) . On the contrary, at histological examination, in the paw biopsies from WT mice (Fig. 2c) after carrageenan administration, marked inflammatory changes were observed including pronounced cellular infiltration (Fig. 2d) .


Figure 2
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  		Figure 2. Effects of a functional PPAR-{alpha} gene on paw injury. Paw biopsies were taken 4 h after injection of carrageenan. Tissue biopsies were with stained with trichrome stain. No histological alterations were observed in the paw tissues collected from sham WT mice (a) and from sham PPAR-{alpha}KO mice (b). On the contrary, marked inflammatory changes (c) were observed including pronounced cellular infiltration (d) in paw tissues collected from WT mice. The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant augmentation of pathological changes in the paw tissues (e) including more inflammatory cell infiltration (f). The figure is representative of at least three experiments performed on different experimental days.

 
Absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant augmentation of pathological changes in the paw tissues (Fig. 2e) including more inflammatory cell infiltration (Fig. 2f) .

The potential role of the PPAR-{alpha} gene on apoptosis in acute inflammation was evaluated by immunohistochemical detection of FasL. No positive staining for FasL was observed in the paw tissues collected from sham WT mice (Fig. 3a ) and from sham PPAR-{alpha}KO mice (Fig. 3b) . At 4 h after carrageenan administration, positive staining for FasL is readily detected in the inflamed paw from WT mice (Fig. 3c) , mainly localized in the inflammatory cells (Fig. 3d) , infiltrated in connective tissues (see arrows). The presence of positive staining for FasL in the inflammatory cells infiltrated in connective tissues (Fig. 3e , see arrows) was increased significantly in the absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice (Fig. 3f) at 4 h after carrageenan administration.


Figure 3
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  		Figure 3. Immunohistochemical localization of FasL in the mice paw. Paw biopsies were taken 4 h after injection of carrageenan. Sections were incubated overnight with anti-FasL antibody (1:500 in PBS, v/v). The counter-stain was developed with DAB (brown color) and nuclear fast red (red background). No positive staining for FasL was observed in the paw tissues collected from sham WT mice (a) and from sham PPAR-{alpha}KO mice (b). At 4 h after carrageenan administration, positive staining for FasL is observed in the paw tissues from WT mice (c), mainly localized in the inflammatory cells (d), infiltrated in connective tissues (see arrows). The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant increase of positive staining for FasL in the inflammatory cells infiltrated in connective tissues (e and f, see arrows). The figure is representative of at least three experiments performed on different experimental days.

 
Effects of the functional PPAR-{alpha} gene on carrageenan-induced pleurisy
To analyze the possible influence of the PPAR-{alpha} receptor during acute inflammation in the lung, we examined the effect of PPAR-{alpha} gene deletion on carrageenan-induced pleurisy, as evaluated at 4 h after carrageenan administration. All WT mice, which had received carrageenan, developed an acute pleurisy, producing turbid exudate (Fig. 4a ). When compared with the number of cells collected from the pleural space of sham WT mice and sham PPAR-{alpha}KO mice, injection of carrageenan to WT mice induced a significant increase in the number of PMNs (Fig. 4b) . The presence of pleural exudate (Fig. 4a) and the number of inflammatory cells (Fig. 4b) in the pleural cavity at 4 h after carrageenan administration were increased significantly in the absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice. The important presence of inflammatory cells in the pleural cavity appeared to be correlated with the influx of leukocytes into the lung tissue. Therefore, we investigate the role of the functional PPAR-{alpha} gene on the neutrophil infiltration by measurement of the activity of MPO, and MPO was elevated significantly (P<0.001) at 4 h after carrageenan administration in WT mice (Fig. 4c) . In PPAR-{alpha}KO mice, lung MPO activity was increased significantly (P<0.01) in comparison with those of WT animals (Fig. 4c) .


Figure 4
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  		Figure 4. Effects of the functional PPAR-{alpha} gene on carrageenan-induced pleural exudate production (a), accumulation of polymorphonuclear cells in pleural cavity (b), and lung neutrophil infiltration (c). Pleurisy was induced by administration of 0.2 ml sterile saline containing 1% {lambda}-carageenan at the level of the left sixth intercostal space. At 4 h, the total volume was recovered by aspiration, and the total exudate was calculated by subtracting the volume injected (2 ml). The leukocytes in the exudate were counted with an optical microscope in a Burker’s chamber after vital trypan blue staining. A significant production of pleural exudate (a) and polymorphonuclear cell infiltration (b) was observed in pleural cavity of WT mice. In addition, the lung tissues of the carrageenan-treated WT mice show a significant increase of MPO activity, the latter being indicative of neutrophil infiltration (c). The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant increase of the presence of pleural exudate (a) and the number of inflammatory cells in the pleural cavity (b) and in the lung (c) at 4 h after carrageenan administration. *, P < 0.01, versus sham; o, P < 0.01, versus carrageenan-WT group.

 
No histological alterations were observed in the lung tissues collected from sham WT mice (Fig. 5a ) and from sham PPAR-{alpha}KO mice (Fig. 5b) . On the contrary, histological examination of lung sections of all WT mice treated with carrageenan showed tissue injury (Fig. 5c) as well as inflammatory cell infiltration (Fig. 5d) . Lung tissues collected 4 h after carrageenan administration from PPAR-{alpha}KO mice showed a significant augmentation of lung injury (Fig. 5e) as well as a significant presence of inflammatory cell (Fig. 5f) .


Figure 5
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  		Figure 5. Effects of the functional PPAR-{alpha} gene on lung injury. Lung biopsies were taken 4 h after injection of carrageenan. Tissues biopsies were stained with H&E. No histological alterations were observed in the lung tissues collected from sham WT mice (a) and from sham PPAR-{alpha}KO mice (b). On the contrary, tissue injury (c) and inflammatory cell infiltration (d) were observed in lung sections of all WT mice treated with carrageenan. The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant augmentation of pathological changes in the lung tissues (e) as well as a significant presence of the inflammatory cell (f). The figure is representative of at least three experiments performed on different experimental days.

 
To test whether the functional PPAR-{alpha} gene may modulate the inflammatory process through the regulation of the secretion of other cytokines, we analyzed the levels of proinflammatory cytokines in PPAR-{alpha}KO and WT. A substantial increase of TNF-{alpha} (Fig. 6a ) and IL-1ß (Fig. 6b) production was found in pleural exudates collected from WT mice at 4 h after carrageenan administration. Pleural exudate production of TNF-{alpha} (Fig. 6a) and IL-1ß (Fig. 6b) was significantly higher in carrageenan-treated, PPAR-{alpha}-deficient mice in comparison with those of WT animals. In agreement with the results observed in the pleural exudates, the lung tissues of the carrageenan-treated WT mice show a significant increase of the TNF-{alpha} (Fig. 6c) and IL-1ß (Fig. 6d) mRNA levels in comparison with the sham WT mice and sham PPAR-{alpha}KO mice. In the lung tissues of carrageenan-treated PPAR-{alpha}KO mice, the TNF-{alpha} (Fig. 6c) and IL-1ß (Fig. 6d) mRNA levels were significantly higher in comparison with those of WT animals measured in the same conditions.


Figure 6
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  		Figure 6. Effects of the functional PPAR-{alpha} gene on pleural exudate production and lung levels of TNF-{alpha} and IL-1ß, where TNF-{alpha} and IL-1ß production was evaluated in the pleural exudates collected at 4 h after carrageenan administration using a colorimetric, commercial ELISA kit and was evaluated in lung tissues using a quantitative real-time PCR. A significant production TNF-{alpha} (a) and IL-1ß (b) was observed in pleural exudates collected from WT mice. The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant increase of pleural exudate production of TNF-{alpha} (a) and IL-1ß (b). Similarly, a significant increase of the TNF-{alpha} (c) and IL-1ß (d) mRNA levels was observed in the lung tissues of the carrageenan-treated WT mice. In the lung tissues of carrageenan-treated PPAR-{alpha}KO mice, the TNF-{alpha} (c) and IL-1ß (d) mRNA levels were significantly higher in comparison with those of PPAR-{alpha} WT animals measured in the same conditions. The results of the quantitative real-time PCR of the TNF-{alpha} and IL-1ß mRNA expression are expressed as a ratio between the number of copies of the target cytokine and the number of copies of the housekeeper (GAPDH) to have an absolute ratio. *, P < 0.01, versus sham; o, P< 0.01, versus carrageenan-WT group.

 
Therefore, we also evaluate the TNF-{alpha} and IL-1ß expression in the lung tissues by immunohistochemical detection. No positive staining for TNF-{alpha} was observed in the lung tissues collected from sham WT mice (Fig. 7a ) and from sham PPAR-{alpha}KO mice (Fig. 7b) . On the contrary, tissue sections obtained from WT animals at 4 h after carrageenan administration demonstrate positive staining for TNF-{alpha} (Fig. 7c) localized in the infiltrated inflammatory cells, pneumocytes, as well as in the vascular wall (Fig. 7d) . In carrageenan-treated PPAR-{alpha}KO mice, the staining for TNF-{alpha} (Fig. 7e) in the infiltrated inflammatory cells, pneumocytes, as well as in the vascular wall (Fig. 7f) was increased visibly and significantly in comparison with the WT mice. Similarly, no positive staining for IL-1ß was observed in the lung tissues collected from sham WT mice (Fig. 8a ) and from sham PPAR-{alpha}KO mice (Fig. 8b) . At 4 h after carrageenan administration, positive staining for IL-1ß, localized in the infiltrated inflammatory cells, pneumocytes, as well as in the vascular wall (Fig. 8c) , was observed in lung tissue sections obtained from WT animals. In carrageenan-treated PPAR-{alpha}KO mice, the staining for IL-1ß (Fig. 8d) in the infiltrated inflammatory cells, pneumocytes, as well as in the vascular wall (Fig. 8e) was increased viisibly and significantly in comparison with the WT mice.


Figure 7
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  		Figure 7. Immunohistochemical localization of TNF-{alpha} in the lung. Lung biopsies were taken 4 h after injection of carrageenan. Sections were incubated overnight with anti-TNF-{alpha} antibody (1:500 in PBS, v/v). The counter-stain was developed with DAB (brown color) and nuclear fast red (red background). No positive staining for TNF-{alpha} was observed in the lung tissues collected from sham WT mice (a) and from sham PPAR-{alpha}KO mice (b). On the contrary, positive staining for TNF-{alpha} (c), mainly localized in the infiltrated inflammatory cells, pneumocytes, as well as in the vascular wall (d), was observed in lung sections from WT animals. The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant increase of the positive staining for TNF-{alpha} (e) in the infiltrated, inflammatory cells, pneumocytes, as well as in the vascular wall (f). The figure is representative of at least three experiments performed on different experimental days.

 

Figure 8
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  		Figure 8. Immunohistochemical localization of IL-1ß in the lung. Lung biopsies were taken 4 h after injection of carrageenan. Sections were incubated overnight with anti-IL-1ß antibody (1:500 in PBS, v/v). The counter-stain was developed with DAB (brown color) and nuclear fast red (red background). No positive staining for IL-1ß was observed in the lung tissues collected from sham WT mice (a) and from sham PPAR-{alpha}KO mice (b). At 4 h after carrageenan administration, positive staining for IL-1ß, localized in the infiltrated inflammatory cells, pneumocytes, as well as in the vascular wall (c), was observed in lung tissue sections obtained from WT animals. The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant increase of the positive staining for IL-1ß (d) in the infiltrated inflammatory cells, pneumocytes, as well as in the vascular wall (e). The figure is representative of at least three experiments performed on different experimental days.

 
The potential effect of the functional PPAR-{alpha} gene on apoptosis in acute lung inflammation was evaluated by immunohistochemical detection of FasL. No positive staining for FasL was observed in the lung tissues from sham WT mice (Fig. 9a ) and from sham PPAR-{alpha}KO mice (Fig. 9b) . At 4 h after carrageenan administration, positive staining for FasL is readily detected in the lung tissues from WT mice (Fig. 9c) , mainly localized in the vascular wall (see arrows) and in pneumocytes (see arrowhead). The presence of positive staining for FasL in the vascular wall (see arrows) and in pneumocytes (see arrowhead) was increased significantly in the absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice (Fig. 9d) .


Figure 9
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  		Figure 9. Immunohistochemical localization of FasL in the lung. Lung biopsies were taken 4 h after injection of carrageenan. Sections were incubated overnight with anti-Fas antibody (1:500 in PBS, v/v). The counter-stain was developed with DAB (brown color) and nuclear fast red (red background). No positive staining for FasL was observed in the lung tissues from sham WT mice (a) and from sham PPAR-{alpha}KO mice (b). Positive staining for FasL (c), mainly localized in the vascular wall (see arrows) and in pneumocytes (see arrowhead), was detected in the lung tissues from WT mice. The absence of a functional PPAR-{alpha} gene in PPAR-{alpha}KO mice resulted in a significant increase of the positive staining for FasL (d) in the vascular wall (see arrows) and in pneumocytes (see arrowhead). The figure is representative of at least three experiments performed on different experimental days.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides the evidence that PPAR-{alpha}-deficient mice have abnormally prolonged responses to acute inflammation. In particular, we have clearly demonstrated that PPAR-{alpha} gene deletion increases the development of carrageenan-induced paw edema, the development of carrageenan-induced pleurisy, the infiltration of the lung and paw with PMN, the degree of proinflammatory cytokine production in the lung, the expression of FasL, and the degree of tissue (paw and lung) injury caused by injection of carrageenan. All of these findings support the view that the PPAR-{alpha} receptor modulates the degree of acute inflammation in the mice.

PPAR-{alpha} expression is relatively high in hepatocytes, heart, enterocytes, muscle, and the kidney [32 ]. PPAR-{alpha} regulates genes involved in the ß-oxidation of fatty acids and lipoprotein metabolism. Various studies have clearly demonstrated, using PPAR-{alpha}-deficient mice, that this receptor is involved in high-density lipoprotein and triglyceride metabolism and in hepatic regulation of apolipoprotein and fatty acid ß-oxidation enzyme expression [33 , 34 ]. The first evidence for a role of PPAR-{alpha} in inflammation was suggested in studies using PPAR-{alpha}KO mice. In particular, it has been shown that the ear inflammation induced by LTB4 or arachidonic acid but not 12-O-tetradecanoylphorbol 13-acetate was prolonged in PPAR-{alpha}KO mice [35 ]. Recently, many PPAR-{alpha} ligands, including the naturally occurring ligand LTB4 and the synthetic ligands fenofibrate and Wy14,643, have been used to investigate the role of the PPAR-{alpha} receptor in inflammation [32 ].

Recently, it has been demonstrated that palmitoylethanolamide reduced inflammatory response by engaging the PPAR-{alpha} nuclear receptor, providing a framework for understanding the biological function of natural fatty acid, which is now identified as an endogenous PPAR-{alpha} ligand [27 ]. In addition, it has been demonstrated that PPAR-{alpha} activators suppress IL-1-induced C-reactive protein (CRP) and IL-6-induced fibrinogen expression, the major acute-phase response (APR) proteins in humans [36 ], whose plasma concentrations are elevated, not only in acute but also in chronic inflammatory states. This anti-inflammatory action of PPAR-{alpha} is not restricted to these genes but applies more generally to other APR genes, such as serum amyloid A and fibrinogen-a and -b [38 ]. PPAR-{alpha} activation leads to a reduction in the formation of nuclear CCAAT/enhancer-binding protein (c/EBP)ß and p50-NF-{kappa}B complexes and thereby reduces CRP promoter activation. Moreover, PPAR-{alpha} increases I{kappa}Ba expression, thus preventing nuclear p50/p65 NF-{kappa}B translocation and arresting their nuclear transcriptional activity. Moreover, chronic treatment with fibrates decreases hepatic C/EBPß and p50-NF-{kappa}B protein expression in mice in a PPAR-{alpha}-dependent manner [37 ]. This latter effect likely 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.

Activation of NF-{kappa}B is crucially involved in FasL expression induced by DNA-damaging agents, such as genotoxic drugs and ultraviolet radiation [38 ]. FasL plays a central role in apoptosis induced by a variety of chemical and physical insults [39 ]. Recently, it has been pointed out that Fas-FasL signaling plays a central role in acute inflammation (e.g., acute lung injury) [39 ]. Furthermore, cell death induced by reactive oxygen species (ROS) depends on FasL expression mediated by redox-sensitive activation of NF-{kappa}B [40 ]. Generation of ROS has been implicated in the induction of cell death and inflammation in the paw and lung tissues after carrageenan administration [41 , 42 ] through NF-{kappa}B activation and expression of FasL. We confirm here that the inflammatory process (carrageenin-induced paw edema and pleurisy) leads to a substantial activation of FasL in the paw and lung tissues, which likely contribute in different capacities to the evolution of acute inflammation. In the present study, we found that the genetic inhibition of PPAR-{alpha} receptors leads to a substantial increase of FasL activation, which also induced a proinflammatory response characterized by a release of IL-1ß and chemokines macrophage-inflammatory protein (MIP)-1{alpha}, MIP-1ß, and MIP-2 [43 ]. There is evidence that the proinflammatory cytokines TNF-{alpha} and IL-1ß help to propagate the extension of a local or systemic inflammatory process [44 45 46 ]. We confirm here that the inflammatory process (carrageenin-induced pleurisy) leads to a substantial increase in the levels of TNF-{alpha} and IL-1ß in the exudates and lung tissues, which likely contribute in different capacities to the evolution of acute inflammation. It is interesting that the levels of these two proinflammatory cytokines are significantly higher in the absence of the functional PPAR-{alpha} gene, suggesting that the PPAR-{alpha} receptor modulates the activation and the subsequent expression of proinflammatory genes. However, in an in vivo study, it has been demonstrated that the treatment of CD-1 mice with fenofibrate or Wy14,643 lead to fivefold higher TNF-{alpha} plasma levels induced by lipopolysaccharide administration as well as to a significantly lower 50% lethal dose than control mice [47 ]. These results were also confirmed in PPAR-{alpha}-deficient mice [47 ]. On the contrary, in the same studies, a modest decrease in TNF expression in peritoneal macrophages from WT mice treated with Wy14,643 has been shown [47 ].

A number of recent studies have demonstrated the importance of specific adhesion molecules in the recruitment of inflammatory cells into an area of inflammation [48 ]. The activation and expression of adhesion molecules allow for the adhesion, conformational change, and extravasations (emigration) of the neutrophil, which may induce local injury and participate in the orchestration of systemic inflammation and all of its consequences. Various studies have clearly demonstrated that PPAR-{alpha} is expressed in various types of human endothelial cells [49 50 51 ], suggesting a role for the PPAR-{alpha} receptor in the down-regulation of endothelial cell inflammatory responses. Moreover, It has been shown that WY14,643 significantly reduced the expression of VCAM-1 in human aortic endothelial cells induced by different in vitro stimuli [49 ]. Similarly, in another study, it has been shown that Wy14,643 or fenofibrate significantly reduced in a time- and concentration-dependent manner that TNF-{alpha} induced VCAM-1 expression in endothelial cells [51 ]. In accordance with these findings, we observed that the absence of a functional PPAR-{alpha} gene led to a significant increase of neutrophil infiltration, suggesting that the PPAR-{alpha} receptor modulates the activation and the subsequent infiltration of neutrophils at inflamed sites.

Further experiments with multiple approaches can be expected to unlock the full potential of modulating the PPAR-{alpha} pathway for therapeutic purposes. Thus, we propose (see Fig. 10 ) the following cycle: inflammation -> NF-{kappa}[INSERT IMAGE]B activation -> FasL expression -> cytokine release -> endothelial injury -> PMN infiltration -> more proinflammatory mediator release (e.g., cytokines) -> organ damage. The PPAR-{alpha} receptor activation would intercept this cycle prior to endothelial injury. The confirmation of this proposed feedback cycle, however, requires further investigation. In conclusion, this study provides evidence that the PPAR-{alpha} pathway modulates the degree of acute inflammation in the mice reducing the FasL activation, the formation of the proinflammatory cytokines, and the neutrophil infiltration.


Figure 10
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  		Figure 10. Proposed scheme of some of the delayed inflammatory pathways in carrageenan-acute inflammation. PPAR-{alpha}, when activated after binding with a specific ligand, interacts with retinoid X receptors (RXR) and regulates the expression of target genes, which are also involved in the acute inflammatory responses. We propose the following cycle: inflammation -> NF-{kappa}B activation -> FasL expression -> cytokine release -> endothelial injury -> PMN infiltration -> more proinflammatory mediator release (e.g., cytokines) -> organ damage. The PPAR-{alpha} receptor activation would intercept this cycle prior to endothelial injury. Thus, the PPAR-{alpha} pathway reduced FasL activation, the formation of the proinflammatory cytokines, and the neutrophil infiltration. See Discussion for further explanations. P, phosphorylation; Ub, ubiquitinization.

 


   ACKNOWLEDGEMENTS
 
This study was supported by a grant from MURST (40%). The authors thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study, Mrs. Caterina Cutrona for secretarial assistance, and Miss Valentina Malvagni for editorial assistance with the manuscript.

Received June 24, 2005; revised January 7, 2006; accepted January 12, 2006.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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