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The Journal of Immunology, 2008, 180, 609 -617
Copyright © 2008 by The American Association of Immunologists, Inc.
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Peroxisome Proliferator-Activated Receptor-{gamma} Ligand, 15-Deoxy-{Delta}12,14-Prostaglandin J2, Reduces Neutrophil Migration via a Nitric Oxide Pathway1

Marcelo H. Napimoga2,*,{dagger}, Silvio M. Vieira{dagger},§, Daniela Dal-Secco{dagger}, Andressa Freitas{dagger}, Fabrício O. Souto{ddagger}, Fabiola L. Mestriner{dagger}, José C. Alves-Filho{dagger}, Renata Grespan{dagger}, Toshihisa Kawai, Sérgio H. Ferreira{dagger} and Fernando Q. Cunha{dagger}

* Laboratory of Molecular Biology, University of Uberaba, Minas Gerais, Brazil; {dagger} Department of Pharmacology {ddagger} Department of Surgery and Anatomy, Faculty of Medicine of Ribeirão Preto, University of São Paulo, São Paulo, Brazil; § Laboratory of Pharmacology, National Institute for Research in the Amazon, Manaus, Brazil; and Department of Immunology, The Forsyth Institute, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ligands for peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}), such as 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ2-mediated activation of PPAR-{gamma} ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ2 administration decreases leukocyte rolling and adhesion to the inflammated mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS–/– mice were not susceptible to 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ2-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-1 expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ2, whereas 15d-PGJ2 inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ2 suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In response to inflammatory signals, cells migrate and accumulate at the sites of inflammation. A multifaceted signaling cascade initiated by the inflammatory insult leads to the activation of neutrophils and production of a vast array of proinflammatory cytokines and chemokines (1, 2, 3, 4, 5). Neutrophils are the main leukocyte subtype participating in innate immune defense against microbial infections, and their migration from blood vessels into tissue is a crucial process for neutrophils to recognize and eliminate the host harmful microorganisms (6). Although exerting a protective role in infection-mediated inflammation, tissue damage is a deleterious consequence of intense neutrophil migration as observed in immune inflammatory diseases (7).

Peroxisome proliferator-activated receptors (PPARs),3 members of the nuclear receptor superfamily (8) have a newly recognized role in inflammation (9, 10). Specifically, a natural PPAR-{gamma} ligand, 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), is produced in a course of inflammatory responses, and suppress the activation of macrophages (11, 12). This suggests that 15d-PGJ2 may play an important role in the regulation of inflammatory reactions in vivo. Several in vitro studies have demonstrated that activation of PPAR-{gamma} by 15d-PGJ2 produces anti-inflammatory effects, such as repression of several inflammatory response genes expression in activated macrophages, including the genes encoding TNF-{alpha}, gelatinase B, and cyclooxygenase 2 (COX-2) (13, 14). In addition, 15d-PGJ2 was found to reduce ischemia/reperfusion injury of the gut by suppressing the infiltration of neutrophils in conjunction with inhibition of TNF-{alpha} and IL-1β production in plasma (15). In line with these findings, treatment with PPAR-{gamma} agonist has been reported to attenuate the inflammatory process of experimental colitis in mice (16, 17) and reduce systemic inflammation in polymicrobial sepsis (18).

Neutrophil adhesion and transmigration are mediated by leukocyte β2 integrins, which interact with immunoglobulins, ICAM-1, ICAM-2, and ICAM-3, and by VLA-4 and {alpha}Vβ3 integrins, which interact with VCAM-1 and PECAM-1, respectively (19). Our group recently demonstrated that NO inhibits neutrophil migration by a mechanism dependent on the expression of ICAM-1 on mesenteric microcirculation vessels of mice subjected to experimental acute peritonitis by an injection of either LPS, carrageenan (Cg), or N-formyl peptide (fMLP) (20). Treatment of experimental peritonitis in mice with chemical inhibitors for NO synthase (NOS) increased the migration of neutrophils into venular endothelium and enhanced the expression of ICAM-1 on the endothelium (20). In connection to PPAR-{gamma} as introduced above, agonists for PPAR-{gamma} appear to amplify inducible NOS (iNOS) expression while they inhibit the activation of the inflammatory signal transduction molecule, NF-{kappa}B (21). Cuzzocrea et al. (22) additionally indicated that rosiglitazone, a chemical agonist of PPAR-{gamma}, reduces cerulean-induced acute pancreatitis by reducing neutrophils migration to the lesion and diminishing the expression of ICAM-1 in the pancreas.

Based on these findings, we hypothesized that 15d-PGJ2-mediated down-regulation of neutrophil migration, may be related to an increase on NO production which, in turn, suppresses ICAM-1 expression on microvessels. Using a mouse model of acute inflammation in mesenteric tissues induced by Cg injection, the migration pattern of neutrophils into the inflammatory lesions and the influence of 15d-PGJ2 on iNOS induction and ICAM expression was evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Adult male C57BL/6 (wild type (WT)) and iNOS-deficient (iNOS–/–) mice weighing 20–25 g were housed in temperature-controlled rooms (22–25°C) with access to water and food ad libitum. All experiments were conducted in accordance with the National Health guidelines for the welfare of experimental animals and with the approval of the Ethics Committee of the Faculty of Medicine of Ribeirão Preto (University of São Paulo, São Paulo, Brazil). The animals were used only in a single experimental group.

Determination of neutrophil migration into peritoneal cavity

WT and iNOS–/– mice were injected s.c. with 15d-PGJ2 (Calbiochem) at 100, 300, and 1000 µg/kg, GW9662 (PPAR-{gamma} antagonist, 1000 µg/kg; Calbiochem), L-NAME (nonspecific inhibitor of NOS, 30 mg/kg; Sigma-Aldrich), aminoguanidine (selective inhibitor of iNOS, 50 mg/kg; Sigma-Aldrich) or rosiglitazone (Avandia, 10 mg/kg; GlaxoSmithKline) respectively, followed by an i.p. injection of Cg (500 µg per cavity; Sigma-Aldrich) 30 min later. Four hours after Cg administration, all animals were euthanized in a CO2 chamber. Cells from the peritoneal cavity were then harvested by washing the cavity with 3 ml of PBS containing 1 mM EDTA, and total counts were performed on a cell counter (Coulter). The percentage of neutrophils in the peritoneal cavity was calculated based on their morphological appearance in a cell smear preparation on glass slide using cytocentrifuge (Cytospin 3; Shandon Lipshaw) and followed by the Rosenfeld staining method. The results are presented as the number of neutrophils per cavity.

Real time in situ microscopic analysis for rolling and adhesion events of neutrophils in the mesenteric microcirculation

Two hours after Cg injection, leukocyte rolling was assessed as previously described (23, 24). In brief, animals were anesthetized and the mesenteric tissue was exposed for microscopic examination in situ. The animals were maintained on a special board thermostatically controlled at 37°C keeping the tissue moist and warm by irrigating with Ringer Locke’s solution (pH 7.2–7.4) containing 1% gelatin. The postcapillary venules, with a diameter of 10–18 µm were chosen and the interaction of leukocytes with the luminal surface of the venular endothelium was evaluated counting the number of rolling leukocytes after 10 min. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for >30 s (25). Cells were counted in the recorded image using five different fields for each animal to avoid variability due to sampling. Data were then averaged for each animal.

Immunofluorescence staining CD54/ICAM-1

WT mice were treated with PBS (0.2 ml; s.c.) or with 15d-PGJ2 at a dose of 1000 µg/kg (s.c.). Thirty minutes later, Cg at a dose of 500 µg/cavity was injected i.p. in the treated animals. After 2 h, the mouse mesenteries were collected, and frozen in vivo by pouring an isopentane-propane mixture (–193°C), cooled in liquid nitrogen, over the living organs. The organs were subsequently freeze-substituted in Tissue-Tek at about –70°C. Mesenteric tissues (5 µm sections) were cut on a cryostat (Leica CM1850). The slides were placed in a humidified chamber, incubated with Triton X-100 for 20 min, and washed with PBS, followed by incubation with glycine (0.1 M). The slides were then washed again with PBS and incubated for 40 min at room temperature with a protein-blocking solution (horse serum, 1:50). All sections were subsequently incubated with fluorescein-conjugated anti-mouse CD54/ICAM-1 Ab (BD Pharmingen) diluted 100 times in PBS-1% BSA for 1 h at 25°C. After extensive washes, slides were fixed with Prolong plus 4',6-diamidino-2-phenylindole (DAPI). All sections were observed under a fluorescence microscope (Leica).

Western blot

For Western blot analysis animals were pretreated with 15d-PGJ2 (1000 µg/kg; s.c.) followed by Cg injection after 15 min. A separate set of mice was injected with aminoguanidine (50 mg/kg) (Sigma-Aldrich), followed by 15d-PGJ2 (1000 µg/kg; s.c.) administration and Cg 15 min later. As controls, animals were injected with Cg (500 µg per animal) or with PBS. Subsequently, the mesenteric tissues were dissected from the animals in each group, and the proteins isolated. Tissue were lysed in 400 µl of buffer (1% Triton X-100, 1 M NaF, 100 mM NaPPi, 1M Na3VO4, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml PMSF) and centrifuged at 4°C for 20 min at 12,300 x g. Equal amounts of protein (90 µg) were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher & Schuell). Molecular weight standard (Bio-Rad) was run in parallel to estimate m.w. Membranes were blocked, overnight at 4°C, in TBS-T (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.1% Tween 20) containing 7% dried milk. After blocking, membranes were incubated, at 4°C overnight, with a rabbit anti-ICAM-1 (1:200) or β-actin (Santa Cruz Biotechnology), used as an internal control (1:1000) diluted in TBS-T containing 7% dried milk. Membranes were then incubated with anti-rabbit IgG conjugated with peroxidase (1/2000) diluted in TBS-T containing 5% dried milk at room temperature for 30 min. Finally, the bands recognized by the specific Ab were visualized using a chemiluminescence-based ECL system (Amersham Biosciences) and exposed to an x-ray film for 30 min (Eastman Kodak). A computer-based imaging system (Gel-Pro Analyzer) was used to measure the intensity of OD of bands.

Detection of cytokines and chemokines by ELISA

Mice received 15d-PGJ2 (1000 µg/kg; s.c.) and after 2 h of Cg stimuli (500 µg per cavity), peritoneal exudates were recovered to cytokine measurements. Levels of TNF-{alpha}, IL-1β, MIP-1{alpha} (CCL3), MIP-2 (CCL-5), and keratinocyte chemokine (KC) (CXCL1) were determined by ELISA using protocols supplied by the manufacturers (R&D Systems) from both experiments. The results are expressed as picograms.

In vitro chemotaxis assay using human neutrophils

To provide a translational aspect of the findings gained from mouse experiments, which determined the effects of 15d-PGJ2 on neutrophils migration, human neutrophils were also used for the in vitro chemotaxis assay. Healthy volunteers were recruited for the use of their blood neutrophils. The study was approved by the Human Subjects Institutional Committee of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, and written informed consent was obtained. Blood neutrophils were isolated from peripheral blood by density-gradient centrifugation using Percoll (Sigma-Aldrich). Neutrophils were washed with HBSS and resuspended in RPMI 1640 medium supplemented with 0.01% BSA (1 x 106 neutrophils/ml) and subjected to pretreatment with 15d-PGJ2 (0.3, 1, and 3 µM), rosiglitazone (10 µM) or with medium alone (negative control) for 2 h at 37°C. Then, neutrophils were centrifuged and washed twice with fresh medium, and incubated with 1400W, cycloheximin, GW9662 or clotrimazole for 30 min at 37°C. Afterward, neutrophils were washed and applied to a chemotaxis assay. Cell migration was assessed by a 48-well microchemotaxis chamber technique. A 28.6-µl aliquot of chemotaxis stimuli (fMLP, 10–7 M) was placed in the lower compartment and 50 µl of cell suspension (1.0 x 106/ml neutrophils) previously treated (as described above) was placed in the upper compartment of the chamber. The two compartments were separated by a polycarbonate filter (5-µm PVP-free polycarbonate filter). The chamber was incubated at 37°C for 1 h. At the end of the incubation period, the filter was removed fixed and stained. The number of migrated cells in five distinct fields was counted by light microscopy after coding the samples. All experiments were repeated at least two times with different cells. The migration was expressed as number of neutrophil per field.

Fluorescence microscopy for F-actin rearrangement in mouse neutrophils

The contents of F-actin residues were determined in mouse neutrophils stimulated with MIP-2 (10–7 M; PeproTech) in the presence or absence of 15d-PGJ2 (3 µM) by fluorescence microscopy. After treatment, neutrophil slides were prepared by cytospin centrifuge, and F-actin was stained with rhodamine-labeled phalloidin (Molecular Probes). Microscopic analysis of fluorescent images was done using an epifluorescence microscope (Olympus BX40-F4) equipped with appropriate filters for rhodamine, and using 100x/1.30 NA oil-immersion objectives. Image capturing was performed with a cooled charged-coupled device CoolSNAP camera (Photometrics). All images were captured using identical camera settings: time of exposure, brightness, contrast and sharpness, and an appropriated white balance set according to the fluorescence filter and acquired and analyzed by Image-Pro Plus 4.0 (Media Cybernetics). The mean fluorescence density was determined from a linear measurement of individual cells’ fluorescence. All cells of at least five randomly chosen fields of each slide, performed in duplicate were analyzed.

Flow cytometry analysis

Peripheral bloods were isolated from mice. After depletion of RBC using ammonium chloride solution, blood leukocytes were resuspended in RPMI 1640 medium supplemented with 10% FBS (Invitrogen Life Technologies), L-glutamine, antibiotics, and 2-ME. Leukocytes (5 x 105/ml) were then preincubated in the presence or absence of 15d-PGJ2 (3 µM) for 3 h in a CO2 incubator at 37°C or stimulated with or without MIP-2 (10–7 M). Leukocytes isolated from the culture were suspended in PBS with 1% BSA and 0.01% sodium azide. The resulting suspension was incubated with anti-CD11a mAb (clone M17/4; eBioscience) or anti-CD11b mAb (clone M1/70; BioLegend), followed by FITC-labeled donkey F(ab')2 anti-rat IgG (Jackson ImmunoResearch Laboratories). Fluorescence data were collected using logarithmic amplification on EPICS Altra flow cytometer (Beckman Coulter). Neutrophil population was gated according to forward- and side-scatter light profile.

Statistical analysis

Data were expressed as mean ± SD. Statistical comparisons between groups were made using one-way ANOVA followed by Bonferroni’s test. Significance was accepted when the value was p ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
15d-PGJ2 decreases neutrophil migration, rolling, and adhesion induced by Cg

Compared with PBS administration, the injection of Cg (500 µg/cavity, i.p.) significantly increased neutrophil migration in mice. In contrast, pretreatment of mice with 15d-PGJ2 (100, 300, and 1000 µg/kg) significantly decreased in a dose-dependent manner the neutrophil migration triggered by Cg injection (Fig. 1A). To clarify the mechanisms by which 15d-PGJ2 activity modulated neutrophil migration to the inflammatory site, we also investigated its effect on leukocyte-endothelium interactions (rolling and adhesion) in mesenteric postcapillary venules. Cg administration increased rolling (Fig. 1B) and adhesion (Fig. 1C) of leukocytes to the endothelium, whereas pretreatment with 15d-PGJ2 significantly decreased such phenomenon (Fig. 1, B and C). Finally, 15d-PGJ2 interestingly inhibited Cg-induced neutrophil migration and adherence, however, not under baseline conditions (Fig. 1, B and C, second column). These results suggest that 15d-PGJ2 activity down-regulates neutrophil-endothelium interactions and consequently neutrophil migration, during the inflammatory process.


Figure 1
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  		FIGURE 1. A, Effects of 15d-PGJ2 on neutrophil migration induced by Cg injection into mouse peritoneal cavity: Mice were pretreated s.c. with 15d-PGJ2 or its respective vehicle (saline with a final concentration of 1% of DMSO), as shown. After 30 min, Cg was injected i.p. (500 µg per cavity). Neutrophil migration was determined 4 h after, and results are expressed as mean ± SD of neutrophil numbers counted in the peritoneal cavity. To evaluate the role of 15d-PGJ2 in leukocyte rolling (B) and adhesion (C) induced by Cg, mice were pretreated s.c. with 15d-PGJ2 or its vehicle. After 30 min, Cg was injected i.p. Leukocyte rolling and adhesion were evaluated by intravital microscopy in the mesentery 2 and 4 h later, respectively. Results are expressed as mean ± SD of 10 animals per group. #, p < 0.05 compared with vehicle treated mice; *, p < 0.05 compared with Cg-injected mice (ANOVA, followed by Bonferroni’s test).

 
Effects of 15d-PGJ2 on Cg-induced ICAM-1 expression on mesenteric microcirculation vessels

Cg injection (500 µg/cavity, i.p.) induced a significant up-regulation of ICAM-1 expression on mesenteric vessel endothelium of mice when compared with i.p. injection of PBS (Fig. 2, A and B). However, pretreatment of mice with 15d-PGJ2 significantly decreased Cg-induced endothelial ICAM-1 expression (Fig. 2C). Western blot analysis additionally showed a Cg up-regulation of ICAM-1 expression (Fig. 2D, lanes 2 and 3) when compared with mice injected with PBS (lane 1), while immunofluorescence data confirmed that 15d-PGJ2 administration decreases Cg-induced ICAM-1 expression (lanes 4 and 5), and this pattern was abrogated in animals pretreated with aminoguanidine, a selective iNOS blocker (lanes 6 and 7). Fig. 2E shows the OD of the Western blot assay for ICAM-1 normalized with β-actin expression.


Figure 2
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  		FIGURE 2. Effects of 15d-PGJ2 on Cg-induced ICAM-1 expression on mesentereric microcirculation vessels. ICAM-1 expression on the microcapillary vessels in mesenteric tissues was determined by fluorescent immunohistochemical staining using anti-ICAM-1-specific mAb conjugated with FITC (A–C). A, Immunofluorescence staining for ICAM-1 in PBS i.p. injected animals. B, Mice treated with PBS (0.2 ml, s.c.) and then injected with Cg (500 µg/cavity). C, Mice treated with 15d-PGJ2 (1000 µg/kg, s.c., 30 min. before) and then injected with Cg. D, ICAM-1 protein expression in mesentery of mice after challenge with Cg in the presence or not of 15d-PGJ2. Mesentery was collected 4 h later, and ICAM-1 protein expression was analyzed by Western blot. E, Intensity of OD of bands measured from Western blots. Density of the ICAM-1 band was normalized to β-actin expression. Protein band intensity is represented as arbitrary units. The results are expressed as mean ± SD of four animals per group. #, p < 0.05 compared with vehicle treated mice; *, p < 0.05 compared with Cg-injected mice.

 
15d-PGJ2 activity does not interfere with Cg-induced release of the neutrophil chemotactic cytokine and chemokines

Next, we investigated the possible interference of 15d-PGJ2 on the release of TNF-{alpha} and IL-1β cytokines, and the release of MIP-1{alpha}, MIP-2, and KC chemokines, in an attempt to clarify whether down-regulation of neutrophil migration promoted by 15d-PGJ2 metabolites is related to a decrease in the release of neutrophil chemotactic mediators. As shown in Fig. 3, mice pretreated with 15d-PGJ2 and challenged with Cg presented similar levels of cytokines and chemokines in the peritoneal exudate, when compared with mice pretreated with PBS and injected with Cg (Fig. 3).


Figure 3
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  		FIGURE 3. 15d-PGJ2 does not interfere with the cytokines and chemokines release. Peritoneal exudate was collected 2 h after challenge with Cg, and the concentration of proinflammatory cytokines TNF-{alpha} (A), IL-1β (B), and chemokines MIP-1{alpha} (C), MIP-2 (D), and KC (E) content was analyzed using ELISA. Results are expressed as means (pg/ml) ± SD of concentration of each factor in exudates (n = 10/group). #, p < 0.05 compared with vehicle treated mice (ANOVA, followed by Bonferroni’s test).

 
15d-PGJ2 activity is dependent on PPAR-{gamma} receptor

Having observed the inhibition of leukocyte recruitment to inflammatory mesenteric tissues in animals pretreated with 15d-PGJ2, we further investigated the direct involvement of PPAR-{gamma} activation in the 15d-PGJ2-mediated suppression of neutrophils migration to the Cg-induced inflammatory site. Although 15d-PGJ2 is known to activate PPAR-{gamma}, it is still unclear whether suppression of neutrophils migration results from the direct activation of PPAR-{gamma} by 15d-PGJ2. As shown in Fig. 4, the injection of Cg (500 µg/cavity, i.p.) induced a significant increase in neutrophil migration into mesenteric postcapillary venules in mice, which was decreased when animals were pretreated with 15d-PGJ2 (1000 µg/kg). Moreover, the group that received a previous administration of GW9662 (PPAR-{gamma} antagonist, 1000 µg/kg) abrogated the inhibitory effect of 15d-PGJ2 on neutrophil migration to inflammatory mesenteric capillary, indicating that 15d-PGJ2 acted directly on PPAR-{gamma}. Importantly, GW9662 treatment did not affect significantly Cg-induced neutrophil migration. Confirming that the activation of PPAR-{gamma} receptor inhibits neutrophil migration we additionally observed that rosiglitazone, a synthetic PPAR-{gamma} agonist, also inhibited Cg-induced neutrophil migration (Fig. 4).


Figure 4
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  		FIGURE 4. Involvement of PPAR-{gamma} activation in 15d-PGJ2-mediated suppression of neutrophil migration. Mice were pretreated s.c. with vehicle or PPAR-{gamma} antagonist GW9662 (1000 µg/kg) 15 min before administration of 15d-PGJ2 or rosiglitazone. Thirty minutes later, animals received an i.p. injection of Cg (500 µg per cavity), and neutrophil migration was determined 4 h later. Results are expressed as mean ± SD of 10 animals per group. #, p < 0.05 compared with vehicle treated mice; *, p < 0.05 compared with Cg-injected mice (ANOVA, followed by Bonferroni’s test).

 
Effects of NO synthesis inhibitors on neutrophil migration in WT and iNOS–/– mice pretreated with 15d-PGJ2

To test our hypothesis that 15d-PGJ2 induces down-regulation of neutrophils migration by increasing NO production which, in turn, suppresses ICAM-1 expression on microvessels, we used several NOS inhibitors. First, injection of Cg (i.p. 500 µg/cavity) caused a significant increase on leukocytes migration, when compared with injection of PBS (i.p.) in WT and iNOS–/– mice (Fig. 5). The pretreatment of the Cg-injected WT mice with L-NAME (nonspecific inhibitor of NOS, 30 mg/kg) or aminoguanidine (selective inhibitor of iNOS, 50 mg/kg) completely abrogated the inhibitory effect of 15d-PGJ2 or rosiglitazone on neutrophil migration (Fig. 5, A and B, respectively), whereas L-NAME or aminoguanidine alone did not affect the increased neutrophils migration induced by Cg injection.


Figure 5
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  		FIGURE 5. Requirement of NO generation in the 15d-PGJ2-mediated suppression of neutrophil migration in response to Cg-triggered inflammation. Mice were pretreated i.p. with vehicle or L-NAME (A) and aminoguanidine (Amino) (B) 15 min before s.c. administration of vehicle, rosiglitazone, or 15d-PGJ2. After 30 min, animals received an i.p. injection of Cg (500 µg per cavity), and neutrophil migration was determined 4 h later. C, WT and iNOS–/– mice were pretreated with 15d-PGJ2 and 30 min later the animals received an i.p. injection of Cg (500 µg per cavity). The neutrophil migration was determined 4 h later. Results are expressed as mean ± SD of 10 animals per group. #, p < 0.05 compared with vehicle treated mice; *, p < 0.05 compared with Cg-injected mice (ANOVA, followed by Bonferroni’s test).

 
NOS are classified into three isoforms, iNOS, endothelial NOS (eNOS), and neuronal NOS (nNOS) (26). Although iNOS gene expression is induced by inflammatory stimuli, eNOS and nNOS genes are constitutively expressed irrespective of stimulation. Therefore, the latter two NOS are also termed as constitutive NOS. Since the mouse model of peritonitis induces an acute inflammation, we examined whether iNOS activation is responsible for the NO-mediated neutrophils migration suppression, using iNOS–/– mice. Very importantly, treatment of iNOS–/– mice with 15d-PGJ2 did not alter the Cg-mediated increase neutrophils migration into mesenteric postcapillary venules as observed in WT mice (Fig. 5C). These findings indicated that NO produced via iNOS activation is associated with the suppression of neutrophils migration caused by 15d-PGJ2.

Neutrophil chemotaxis

We next investigated the influence of 15d-PGJ2 on the in vitro chemotaxis activity of human neutrophils toward fMLP. Fig. 6A shows that fMLP (10–7 M) induced neutrophil migration; however, when neutrophils were preincubated with 15d-PGJ2, neutrophil migration was decreased in a dose-dependent manner, until 3 µM of 15d-PGJ2 reached a significant decrease neutrophil migration (p < 0.05). The same pattern was observed when cells were incubated in the presence of rosiglitazone (10 µM). The suppression of neutrophils migration was not due to the toxic effects of the chemicals, because even in the presence of the highest dose tested, 99% of the cells were viable (trypan blue exclusion assay, data not shown). Interestingly, postincubation of cells with GW9662 (PPAR-{gamma} antagonist) or 1400W (iNOS inhibitor), showed a complete abrogation of the inhibitory effects induced by preincubation of cells with 15d-PGJ2 (Fig. 6, B and C, respectively).


Figure 6
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  		FIGURE 6. Requirement of NO in the 15d-PGJ2-mediated inhibitory effect on human peripheral blood neutrophil migration. Chemotaxis of human neutrophils in response to fMLP (10–7 M) was measured in a chemotaxis chamber. A, Dose response of 15d-PGJ2.Neutrophils were incubated with (B) 15d-PGJ2 or rosiglitazone and GW9662, (C) 15d-PGJ2 and 1400W, (D) 15d-PGJ2 and cyclohexamide and (E) 15d-PGJ2 or rosiglitazone and clotrimazole. Data are means ± SD. #, p < 0.05 compared with vehicle treated neutrophil; *, p < 0.05 compared with fMLP-stimulated neutrophils (ANOVA, followed by Bonferroni’s test).

 
It was further investigated whether 15d-PGJ2-mediated suppression of neutrophils migration is associated with the interference of new protein synthesis by neutrophils in vitro. We used a chemical cyclohexamide that acts at the large subunit of the ribosome and inhibits mRNA-protein translation. The ability of fMLP to increase neutrophil chemotaxis, whether preincubated or not in the presence of 15d-PGJ2,was not statistically affected by cyclohexamide (Fig. 6D). In addition, neutrophils incubated with 15d-PGJ2 and rosiglitazone followed by incubation with clotrimazole (a potassium channel blocker) demonstrated that neutrophils chemotaxis is dependent on K+ channels (Fig. 6E).

Actin polymerization in the presence of 15d-PGJ2

During migration, changes in actin cytoskeleton enable cells to move toward chemoattractant factors. In response to external chemoattractant stimuli, intracellular signals induce a dynamic remodeling of the cytoskeleton, which results in cell shape changes and increases their motility (27, 28). Specially, the fundamental mechanical changes of cytoskeleton remodeling derive from rearrangement of filamentous action (F-actin), which does not require new protein translation (synthesis). We investigated the alterations of F-actin contents in neutrophils in response to stimulation with MIP-2 in the presence or absence of 15d-PGJ2. Fig. 7 shows that after treatment with MIP-2, a significant increase on F-actin polymerization was observed when compared with neutrophils under basal conditions (Fig. 7, A and B). However, neutrophils preincubated with 15d-PGJ2 and stimulated with MIP-2 showed a decrease on F-actin rearrangement very similar to the F-actin rearrangement at basal levels (Fig. 7C). In addition, treatment with 15d-PGJ2 alone did not induce any expression of F-actin when compared with basal conditions (Fig. 7D). Differences in intensity of fluorescence among all treatments are demonstrated in Fig. 7E. These data indicated that 15d-PGJ2-mediated suppression of neutrophils migration may be associated with diminished cytoskeletal F-actin rearrangement.


Figure 7
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  		FIGURE 7. 15d-PGJ2 decreases F-actin polymerization of mouse neutrophils stimulated with MIP-2. The contents of F-actin in mouse blood neutrophils were analyzed by fluorescence microscopy. Neutrophils were incubated with medium alone (A), MIP-2 (B), 15d-PGJ2 followed by MIP-2 stimulation (C), or 15d-PGJ2 (D). F-actin filaments were stained with rhodamine-phalloidin. Panels show images representative of at least three independent experiments. In addition, cells were imaged and fluorescence intensity of F-actin quantified. Data represent mean ± SD from three independent experiments. #, p < 0.05 compared with medium alone. *, p < 0.05 compared with cells incubated with MIP-2 (ANOVA followed by Bonferroni).

 
Neutrophil cellular adhesion molecule expression in the presence of 15d-PGJ2

The CD11a/CD18 and CD11b/CD18 are expressed on the surface of most leukocytes and interacts with ICAM-1 and ICAM-2 on endothelial cells to cause firm adhesion (29). Thus, we analyzed the expression of this molecule to observe whether 15d-PGJ2 or MIP-2 may influence the expression of these cellular adhesion molecules on neutrophils. Data demonstrated no significant alteration on surface expression of CD11a or CD11b in the presence of 15d-PGJ2 or MIP-2 when compared with neutrophils incubated with medium alone (Fig. 8). These data indicated that 15d-PGJ2-mediated suppression of neutrophils migration is not associated with decreased cellular adhesion molecules expression by neutrophils.


Figure 8
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  		FIGURE 8. Flow cytometry assay. Representative flow cytometry graph of isolated mice neutrophils stained with anti-CD11a in the presence of medium alone (A) or stimulated with 3 µM 15d-PGJ2 (C), or 100 ng/ml MIP-2 (E). Neutrophils stained with anti-CD11b in the presence of medium alone (B) or stimulated with 15d-PGJ2 (D), or MIP-2 (F).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we partially elucidated the molecular mechanism underlying the role of PPAR-{gamma}, and 15d-PGJ2-mediated suppression of neutrophils migration toward inflammatory stimuli using a mouse peritonitis model, in which neutrophil migration is a key event. Furthermore, we also showed that this decreased neutrophil migration is associated with 15d-PGJ2-mediated suppression of ICAM-1 expression on endothelial cells, but it is not linked to decreased cytokines/chemokines production or CD11a expression by neutrophils. Moreover, its action is dependent on NO signaling pathway and also by decreased F-actin polymerization in neutrophils.

Recent research findings suggest that a novel group of antidiabetic agents, PPAR-{gamma} agonist chemicals, exhibit anti-inflammatory properties in the vessel wall (30). These agents were originally found to diminish blood glucose levels by increasing insulin sensitivity in peripheral organs and influence lipid metabolism in treated patients. Since it was previously shown that endothelial cells (EC) express PPARs (31), accumulating interests were focused on the anti-inflammatory effects of PPAR agonists on the vascular tissue. In addition, it was revealed that the activation of PPAR-{gamma} with its agonists effectively attenuates inflammatory processes in a number of organs such as heart (32, 33, 34, 35), kidney (36, 37), lung (38), and intestine (15, 39) against ischemia-reperfusion injury.

It has been demonstrated that endogenous production of cyclopentenone prostaglandins including 15d-PGJ2 occurs via cyclooxygenase 2 activation during inflammation and may favor the resolution of the inflammatory response (40). Urinary 15d-PGJ2 has also been detected in healthy volunteers in a range of 6 to 7 pg/mg creatinine (41). Despite this evidence, we are providing new evidence that exogenously administration of 15d-PGJ2 could be useful to suppress inflammatory injury mediated by neutrophils migration.

A key step in the inflammatory response is the recruitment of neutrophils from the blood into infectious or injured tissue (42). Although, in this study, the production of proinflammatory cytokines IL-1β and TNF-{alpha} and chemokines, in the peritoneal exudates was not altered by the 15d-PGJ2 treatment, the down- regulation of neutrophil migration by 15d-PGJ2 reduces the inflammatory outcomes in blood vessels. Neutrophil recruitment is a complex process that involves a sequence of molecular-mechanical events on leukocytes and endothelial cells; 1) rolling, 2) arrest, 3) firm adhesion, and 4) trans-endothelial diapedesis that depends on distinct cell-cell adhesion molecules and cytokines/chemokines production (43). It was previously demonstrated that, among a number of PPAR activators, 15d-PGJ2 specially affects leukocyte interaction with ECs, partly through preventing up-regulation of VCAM-1 and E-selectin by ECs in response to inflammatory stimuli (44). However, until the present study, it was still unclear whether PPAR-{gamma} activator was able to affect the expression of ICAM-1 by ECs. Our data suggests that 15d-PGJ2 suppresses the expression of not only VCAM-1 and selectin but also ICAM-1 by the vascular endothelium, which will lead to down-regulation the neutrophil rolling, firm adhesion, and transmigration onto the endothelium. In contrast, the heterodimer CD11a/CD18 expression on the surface of most leukocytes, and responsible to interact with ICAM-1 and ICAM-2 on endothelial cells to cause firm adhesion (29) was not altered on neutrophils after 15d-PGJ2 treatment.

An increasing body of in vivo evidence shows that 15d-PGJ2 possesses anti-inflammatory properties (18, 45, 46). We demonstrated that 15d-PGJ2-mediated inhibitory effect on neutrophils migration was completely abrogated by chemical inhibitors of NO synthesis, suggesting a possible role of NO on this process. Despite the findings showing a diminished neutrophils migration as a consequence of reduced NO production, the ability of NO derived from both iNOS and eNOS to down-regulate neutrophil migration by inhibiting ICAM-1 to an inflammatory site was reported by us and other groups (47, 48). Pharmacological inhibition of both eNOS and iNOS or iNOS gene deletion leads to an increase in leukocyte trans-endothelium migration and most of these effects displayed by NO are mediated by cGMP, derived from sGC activation (20, 48, 49, 50). Back to the results of the present study, since iNOS gene knockout mice lost the susceptibility to 15d-PGJ2-mediated suppression of neutrophils migration to inflammatory mesenteric tissues, NO produced by iNOS activation appeared to be responsible for this 15d-PGJ2-mediated suppression of neutrophils migration.

Besides its in vivo inhibitory effect on neutrophils migration, 15d-PGJ2 also demonstrated direct inhibitory effects on neutrophil chemotactic activity in vitro, which was dependent on NO production. However, this effect was not reversed by cyclohexamide treatment, indicating that this inhibition effect is not dependent on protein synthesis. In fact, there is evidence that 15d-PGJ2 is able to increase NO release in endothelial cells without increasing the expression of eNOS (51). In agreement, we recently demonstrated that NO directly inhibited neutrophil chemotaxis in vitro (52). Our pharmacological experiments with K+ channel inhibitors support this possibility and identify calcium-activated K+ channels as downstream components of the nongenomic PPAR-{gamma} signaling pathway implicated in the regulation of neutrophil functions. In addition, recent evidences of the literature suggest that PPAR agonists also act via a nongenomic pathway that opens this calcium operated K+ channels to promote rapid broad-spectrum analgesia (53). Although the molecular steps that link PPAR-{gamma} activation to the gating of K+ channels remains undefined, our results raise the possibility that it could be mediated by an increase of NO production. As a manner of fact, there is strong evidence that NO is able to activate, directly or indirectly, K+ channels (54, 55).

In conclusion, as we hypothesized, PPAR-{gamma} ligand 15d-PGJ2 suppressed neutrophils migration in a NO expression dependent manner via iNOS activation. More specifically, 15d-PGJ2 reduces neutrophil migration to the acute inflammatory mesenteric lesion as a result of reduction of rolling and adhesion activities of neutrophils to endothelial cells. Such reduction of neutrophils migration required NO expression via iNOS activation. Furthermore, diminished rolling and adhesion activities of neutrophils to endothelial cells were supported by inhibitory effects of 15d-PGJ2 on ICAM-1 expression by endothelial cells, but not to any alterations on the expression of the cellular adhesion molecules (CD11a/CD11b). It was also demonstrated that 15d-PGJ2 inhibits F-actin polymerization, which is required for their fundamental movement. Taken together, our results suggest that exogenous application of 15d-PGJ2 may be able to suppress the development of acute inflammatory lesions, which are initiated by neutrophil recruitment.


   Acknowledgments
 
We thank Dr. Marcos A. Rossi, Monica Azevedo de Abreu, and Giuliana Bertozi Francisco.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo Grant 05/60295-8, Fundaçao de Amparo à Pesquisa do Estado de Minas Gerais Grant 34/07, and National Institute of Dental and Craniofacial Research Grant DE 18310. Back

2 Address correspondence and reprint requests to Dr. Marcelo H. Napimoga, Laboratory of Molecular Biology, University of Uberaba, Avenue Nenê Sabino, 1801, Uberaba, Minas Gerais 38055-500, Brazil. E-mail address: marcelo.napimoga{at}uniube.br Back

3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; 15d-PGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; COX, cyclooxygenase; NOS, NO synthase; iNOS, inducible NOS; Cg, carrageenan; WT, wild type; DAPI, 4',6-diamidino-2-phenylindole; eNOS, endothelial NOS; nNOS, neuronal NOS; EC, endothelial cell; KC, keratinocyte chemokine. Back

Received for publication June 21, 2007. Accepted for publication October 16, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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