1 15-Deoxy-12 , 14-prostaglandin J 2 : A prostaglandin D 2 metabolite generated during inflammatory processes

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J(2) series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), which have been suggested to exert anti-inflammatory effects in vivo. Meanwhile, the mechanism of these effects is not well understood and the natural site and the extent of its production in vivo remain unclear. In the present study, we raised a monoclonal antibody specific to 15d-PGJ(2) and determined its production in inflammation-related events. The monoclonal antibody (mAb11G2) was raised against the 15d-PGJ(2)-keyhole limpet hemocyanin conjugate and was found to recognize free 15d-PGJ(2) specifically. The presence of 15d-PGJ(2) in vivo was immunohistochemically verified in the cytoplasm of most of the foamy macrophages in human atherosclerotic plaques. In addition, the immunostaining of lipopolysaccharide-stimulated RAW264.7 macrophages with mAb11G2 demonstrated an enhanced intracellular accumulation of 15d-PGJ(2), suggesting that the PGD(2) metabolic pathway, generating the anti-inflammatory PGs, is indeed utilized in the cells during inflammation. The activation of macrophages also resulted in the extracellular production of PGD(2), which was associated with a significant increase in the extracellular 15d-PGJ(2) levels, and the extracellular 15d-PGJ(2) production was reproduced by incubating PGD(2) in a cell-free medium and in phosphate-buffered saline. Moreover, using a chiral high performance liquid chromatography method for separation of PGD(2) metabolites, we established a novel metabolic pathway, in which PGD(2) is converted to 15d-PGJ(2) via an albumin-independent mechanism.

The prostaglandins (PGs) are a family of structurally related molecules that are produced by cells in response to a variety of extrinsic stimuli and regulate cellular growth, differentiation and homeostasis (1,2).PGs are derived from fatty acids, primarily arachidonate, which are released from membrane phospholipids by the action of phospholipases.Arachidonate is first converted to an unstable endoperoxide intermediate by cyclooxygenases and subsequently converted to one of several related products, including PGD 2 , PGE 2 , PGF 2α , prostacyclin (PGI 2 ), and thromboxane A 2 , through the action of specific PG synthetases.PGD 2 , among them, is a major cyclooxygenase (COX) product in a variety of tissues and cells and has marked effects on a number of biological processes, including platelet aggregation, relaxation of vascular and nonvascular smooth muscles and nerve cell functions (3).The PGs are physiologically present in body fluids in picomolar-tonanomolar concentrations (4); however, arachidonate metabolism is highly increased under several pathological conditions, including hyperthermia, infection and inflammation (5), and local PG concentrations in the micromolar range have been detected at sites of acute inflammation (6).In vitro, PGD 2 spontaneously converts into the cyclopentenone PGs of the J series, such as PGJ 2 , ∆ 12 -PGJ 2 , and 15-deoxy-∆ 12,14 -PGJ 2 (15d-PGJ 2 ) (Fig. 1) (7,8).It is not known whether this pathway is utilized in the organism, but it is clear that J 2 prostanoids are synthesized in vivo.This is based on the observations that ∆ 12 -PGJ 2 is a natural component of human body fluids (9) and that ∆ 12 -PGJ 2 synthesis is suppressed by treatment with COX inhibitors (9).The natural precursor of PGJ 2 derivatives appears to be PGD 2 , because its in vivo administration leads to a large increase in ∆ 12 -PGJ 2 (9).
The cyclopentenone PGs have been reported to have their own unique spectrum of biological effects, including inhibition of macrophage-derived cytokine production (10,11) and IκB kinase (12,13), induction of synoviocyte and endothelial cell apoptosis (14), induction of glutathione S-transferase gene expression (15) and intracellular oxidative stress (16), and potentiation of apoptosis in activated macrophages (17).Furthermore, recent studies have reported that they could function as a feedback regulator of the inflammatory response (12,13).However, the mechanism of these effects is not well understood, and the natural site and the extent of their productions in vivo remain unclear.Because PGD 2 is the major prostaglandin in most tissues (4), it is likely that the cyclopentenone-type PGD 2 For determination of PGD 2 , a solid phase enzyme immunoassay (Cayman Chemical) was performed as suggested by the manufacturer, and the PGD 2 level was determined using a standard curve and a linear log-logit transformation.For determination of 15d-PGJ 2 in the cell culture medium, the medium (5 ml) was extracted immediately with 3 x 5 ml of ethyl actetate, and the solvent was evaporated under nitrogen.µl aliquot of the competitor/antibody mixture was added to each well and incubated for 2 h at 37 O C. After discarding the supernatants and washing three times with PBS/Tween, the second antibody was added, and the enzyme-linked antibody bound to the well was revealed as previously described.The 15d-PGJ 2 levels in the medium were determined using a standard curve and a linear log-logit transformation.For determination of 15d-PGJ 2 upon in vitro incubation of PGD 2 in cell-free medium or PBS, an aliquot (200 µl) of the reaction mixtures was directly subjected to the competitive ELISA assay.

Immunochemical Detection of 15d-PGJ2 in Human Atherosclerotic
Lesions.Aortic wall samples were obtained at autopsy from five cases of arterial atherosclerosis without diabetes mellitus or any other arterial disorders, performed after their family members granted informed consent.Tissue samples of each case were processed for making frozen materials and used for hematoxylin-eosin stain and immunohistochemical stain.The samples were embedded in OCT compound TM (Sakura Fine Technical Co., Tokyo, Japan), stored at -80 O C, and cut into 6-mm-thick sections by a cryostat.The sections were rehydrated in distilled water, quenched with 3% hydrogen peroxide for 15 min at 4°C, rinsed in PBS, and pretreated with 3% nonimmune serum followed by blocking endogenous avidin/biotin activity using a kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions.
The sections were then incubated overnight at 4°C with the primary antibodies.
Immunoreaction was visualized by the avidin-biotin-immunoperoxidase complex method using the appropriate Vectastain ABC kit (Vector).Immunostained sections were counterstained with hematoxylin.Sections from which the primary antibodies were omitted served as negative reaction controls.

RESULTS
Monoclonal Antibody Specific to 15d-PGJ 2 .15d-PGJ 2 is believed to be physiologically formed through the non-enzymatic conversion of PGD 2 but has never been definitively proven to exist in vivo.In view of its biological significance, it is critical to provide evidence that 15d-PGJ 2 is endogenously produced in vivo.To this end, we produced a monoclonal antibody directed to 15d-PGJ 2 .The antibody was raised against the 15d-PGJ 2 -KLH conjugate, which was prepared from the reaction of KLH with 15d-PGJ 2 in the presence of EDC and sulfo-NHS (Fig. 2A).During the preparation of the monoclonal antibodies, hybridomas were selected on the basis of the ability of their antibodies to bind to the 15d-PGJ 2 -BSA conjugate.After repeated screening, three clones were obtained.Among them, the antibody produced by the clone 11G2 most strongly recognized the 15d-PGJ 2 -BSA conjugate but did not recognize the native BSA (Fig. 2B).The antibody was then tested for immunoreactivity with PGs.As shown in Fig. 2C produced within the cells was stained with mAb11G2.As shown in Fig. 4A and 4B (panels a and b), LPS led to a significant induction of COX-2 in RAW264.7 macrophages.Consistent with the COX-2 up-regulation, exposure of the cells to LPS resulted in the appearance of 15d-PGJ 2 immunoreactivity in essentially all cells (Fig. 4B, panels c and d).An immunofluorescence double-labeling of the activated cells revealed almost identical cellular distribution of COX-2 and 15d-PGJ 2 (Fig. 4C).These results were consistent with the in vivo observations that 15d-PGJ 2 was detected intracellularly in the macrophage-derived foam cells in atherosclerotic lesions (Fig. 3).Taken these in vivo and in vitro data together, it is evident that the production of 15d-PGJ 2 , is highly accelerated in the cells during inflammatory processes.

Extracellular Production of 15d-PGJ 2 in the Activated RAW264.7 Macrophages.
PGs are synthesized in a broad range of tissue types and serve not only as autocrine but also as paracrine mediators to signal changes within the immediate environment.Hortelano et al. (17) have also suggested that 15d-PGJ 2 may contribute to the resolution of inflammation as a paracrine factor.The extracellular production of 15d-PGJ 2 in the activated macrophages was indeed suggested by the observation that the culture medium of macrophages treated with LPS exerted an excitotoxic effect on neurons (data not shown).To examine whether 15d-PGJ 2 is extracellularly produced during inflammation, the levels of 15d-PGJ 2 in the culture medium of the activated macrophages were measured by a competitive ELISA assay.As shown in Fig. 5A, the calibration range (0.1 -10 nmol) of the standard curve for 15d-PGJ 2 was obtained.In parallel with the COX-2 up-regulation, the extracellular levels of PGD 2 in the LPS-stimulated RAW264.7 macrophages were significantly increased (Fig. 5B).In addition, accompanied by by guest on September 16, 2017 http://www.jbc.org/Downloaded from the production of PGD 2 , a significant amount of 15d-PGJ 2 was accumulated in the culture medium (Fig. 5C).The levels of these PGs reached maximums after 12 h of incubation and then decreased thereafter.These data proved that 15d-PGJ 2 was produced extracellularly during inflammatory processes.

Extracellular Conversion of PGD 2 to 15d-PGJ 2 in Cell-Free Medium and in
Phosphate-buffered Saline.Because PGD 2 is known to be converted sequentially to the J 2 derivatives of PGs in vitro (Fig. 1) (21), it was anticipated that 15d-PGJ 2 could be produced extracellularly in the medium through the metabolism of PGD 2 .To examine the extracellular conversion of PGD 2 to 15d-PGJ 2 , 1 mM PGD 2 was incubated in the cell-free medium and the formation of 15d-PGJ 2 was examined.As shown in Fig. 6A, 15d-PGJ 2 was detectable by the ELISA assay within 1 h after initiating the incubation.The concentration of 15d-PGJ 2 increased almost linearly from 0 -8 h and reached a maximum concentration at 24 h.The amount corresponded to the 8% PGD 2 that disappeared during incubation.The maximum levels were maintained for the duration of the experiment.
Serum albumin has been identified as the plasma protein which can catalyze the in vitro transformation of PGD 2 into the J 2 derivatives of PGs in aqueous buffer (7).Accordingly, to examine whether serum albumin contained in the medium functioned as a catalyst in the conversion of PGD 2 to 15d-PGJ 2 , 1 mM PGD 2 was incubated in PBS containing 10 mg/ml of human serum albumin.As expected, PGD 2 was similarly converted to 15d-PGJ 2 in PBS containing serum albumin (Fig. 6B).However, to our surprise, the conversion was dramatically accelerated in the absence of albumin.These data suggest that serum albumin may be rather inhibitory toward the production of 15d-PGJ 2 from PGD 2 .
A Novel PGD 2 Metabolic Pathway.The data (Fig. 6B) contradict the mechanism of PGD 2 metabolism (Fig. 1), in which PGD 2 is sequentially converted to PGJ 2 , ∆12 -PGJ 2 , and 15d-PGJ 2 and the route of conversion leading from PGJ 2 to ∆ 12 -PGJ 2 is catalyzed by serum albumin, therefore suggesting the presence of an albumin-independent mechanism for production of 15d-PGJ 2 .To establish a mechanism of transformation of PGD 2 into the cyclopentenone-type PGJ 2 derivatives, we developed a chiral HPLC method for separation of 12 PGD 2 metabolites and investigated the conventional PGD 2 metabolic pathway in detail.When PGD 2 (1 mM) was incubated in PBS for 24 h, three products (a, b, and c) were mainly detected (Fig. 7A).Over the course of the 24 h period of incubation, no additional abundant products were formed and there were only minor changes in the product pattern (data not shown).The UV spectra of these products were almost indistinguishable.Based on the identical retention time and co-chromatography with authentic PGs, the products a, b, and c were suggested to be PGJ Serum albumin has been previously identified as the endogenous catalyst of PGD 2 metabolism (7-9).In addition, these workers have identified ∆ 12 -PGJ 2 as the major product in the albumin-catalyzed PGD 2 metabolism.Hence, to characterize the albumin-dependent PGD 2 metabolic pathway, PGD 2 was incubated in PBS containing human serum albumin (10 mg/ml) at 37 O C, and the products were analyzed by chiral-phase HPLC.As shown in Fig. 8A (upper chromatogram), incubation of PGD 2 with human serum albumin yielded, not only the same products (15d-PGD 2 , PGJ 2 , and 15d-PGJ 2 ) as those detected in the spontaneous conversion of PGD 2 in albumin-free solution (Fig. 7A), but also a major product which cochromatographed on HPLC with ∆

DISCUSSION
Immunochemical detection is a powerful tool that can be used to evaluate the presence of a desired target and its subcellular localization.The major advantages of this technique over other biochemical approaches are the evaluation of small numbers of cells or archival tissues that may otherwise not be subject to analysis.In this study, we obtained a murine monoclonal antibody, mAb11G2, that clearly distinguished the 15d-PGJ 2 -protein conjugate from the native protein.Characterization of the antibody revealed that the monoclonal antibody was directed almost exclusively against free 15d-PGJ 2 (Fig. 2).It was expected that mAb11G2 would be useful in assessing the endogenous production of 15d-PGJ 2 in response to inflammatory stimuli.In vivo detection of 15d-PGJ 2 using mAb11G2 was first attempted in the tissue samples from the patients with atherosclerosis.Atherosclerosis is considered to be a form of chronic inflammation resulting from interaction between modified lipoproteins, monocyte-derived macrophages, T cells, and the normal cellular elements of the arterial wall.
This inflammatory process can ultimately lead to the development of complex lesions, or plaques, that protrude into the arterial lumen.In the present study, we confirmed that atheromatous lesions indeed contained high levels of COX-2, colocalizing mainly with foamy macrophages (Fig. 3C).In addition, 15d-PGJ 2 was also found to localize predominantly with the lesional macrophages (Fig. 3D).These observations raise the possibility that 15d-PGJ 2 may play a role in the pathogenesis of inflammation-related disorders, such as atherosclerosis.
Activation of the host immune system by Gram-negative bacteria can be reproduced in vitro by incubation of cells with LPS and pro-inflammatory cytokines.Macrophages participate actively in the onset of inflammation and immune system activation by releasing cytokines that amplify the initial inflammatory stimulation, bioactive lipids (e.g., PGs and leukotrienes), reactive oxygen species and reactive nitrogen species that exert cytotoxic effects against pathogens and tumor cells (22)(23)(24)(25).As result of activation, macrophages express pro-inflammatory enzymes, such as COX-2, nitric oxide synthase-2, and matrix metalloproteinases (25) and produce an array of prostanoids, including PGE 2 and thromboxane A 2 (26), considered the more atherogenic eicosanoids.In the present study, we by guest on September 16, 2017 http://www.jbc.org/Downloaded from provided evidence that the production of 15d-PGJ 2 was augmented in the activated RAW264.7 macrophages with LPS in vitro (Fig. 4).These data coincided with the observation that the 15d-PGJ 2 synthesis was predominantly enhanced in the macrophagederived foam cells in vivo (Fig. 3).It is suggested that the enhanced production of 15d-PGJ 2 in the activated macrophages may represent an inflammatory response, which contributes to negative regulation of inflammation.Recent studies have shown that 15d-PGJ 2 is a highaffinity ligand for peroxisome proliferator-activated receptor γ (PPARγ) (27,28).15d-PGJ 2 represses several genes in activated macrophages, including the inducible nitric oxide synthase and tumor necrosis factor α genes, and this repression is suggested to be at least partly dependent on PPARγ expression (10,11,29).The anti-inflammatory effect of 15d-PGJ 2 has also been deduced from models of carrageenin-induced inflammation in which a twophase PG release has been described after expression of COX-2: PGE 2 synthesis predominates during the early inflammatory step, whereas 15d-PGJ 2 substitutes PGE 2 formation at the end of the process, coincident with the accumulation of macrophages (30,31).
Therefore, the physiopathological relevance of the enhanced synthesis of 15d-PGJ 2 in activated macrophages may represent a mechanism in which 15d-PGJ 2 functions as a feedback regulator of the inflammatory reponses (31).
On the other hand, recent studies have shown that 15d-PGJ 2 directly inhibits the NF-κB-dependent gene expression through covalent modifications of critical cysteine residues in IκB kinase and the DNA-binding domains of NF-κB subunits (12,13), leading to the PPARγindependent resolution of inflammation.Several previous observations have also indicated that PGD 2 and its J-ring metabolites might exert effects through interactions with intracellular proteins: (i) Narumiya et al. (32) have shown that radiolabeled ∆ 12 -PGJ 2 is actively incorporated into cells and transferred to the nucleus, where it is associated with proteins; (ii) some PGs, including PGD 2 , PGJ 2 , and ∆ 12 -PGJ 2 , have been shown to bind with high affinity to liver fatty acid-binding protein and intracellular protein involved in the uptake, intracellular transport, and metabolism of free fatty acids and their acyl-CoA esters (33).The A and J series of PGs are characterized by the presence of a cyclopentenone ring system that contains an electrophilic carbon that can react covalently by means of the Michael addition reaction with nucleophiles, such as the free sulfhydryls of glutathione and cysteine residues in cellular human atherosclerotic plaques.In addition, the intracellular production of 15d-PGJ 2 was observed in the activated RAW264.7 macrophages with LPS.We also observed the accumulation of 15d-PGJ 2 in the culture medium of activated macrophages and the spontaneous conversion of PGD 2 to 15d-PGJ 2 in the cell-free medium.Thus, 15d-PGJ 2 was produced not only intracellularly but also extracellularly via nonenzymatic conversion of  Competitors: , arachidonate; , 15d-PGJ 2 ; , ∆ 12 -PGJ 2 ; , PGD 2 ; , 15d-PGD 2 ; , PGJ 2 .

Fig. 4 .
Fig. 4. Intracellular production of 15d-PGJ 2 in the activated RAW264.7 macrophages.(A) Immunoblot analysis of COX-2 in RAW264.7 macrophages exposed to LPS. (B) Immunocytochemical detection of COX-2 (panels a and b) and 15d-PGJ 2 (panels c and d) in the activated RAW264.7 macrophages.The cells were exposed to 10 µg/ml LPS for 24 h at 37 O C. Fluorescein isothiocyanate fluorescence (COX-2, green) is shown in the upper panels (a and b); Cy TM 3 fluorescence (15d-PGJ 2 , red) is shown in the lower panels (c and d).(C) An immunofluorescence double-labeling of activated RAW264.7 macrophages.The cells were exposed to 10 µg/ml LPS for 24 h at 37 O C.

Intracellular Accumulation of 15d-PGJ 2 in Human Atherosclerotic Lesions. We
Downloaded fromdisplayed no significant 15-PGJ 2 immunoreactivity.No immunoreaction product deposits were detected in sections with omission of the primary antibodies (data not shown).These observations verified for the first time the intracellular accumulation of 15d-PGJ 2 in vivo.
, mAb11G2 recognized 15d-PGJ 2 most significantly.Both PGJ 2 and ∆ 12 -PGJ 2 also served as weak antigens, although their inhibitions were about 50% times lower than 15d-PGJ 2 .The antibody did not cross-react with other PGs, such as PGA 2 , PGB 2 , PGD 2 , PGE 2 , PGF 2α , and PGI 2 (data not shown).Taken together, these data indicated that mAb11G2 was directed almost exclusively against the structure of 15d-PGJ 2 .firstexamined the in vivo presence of 15d-PGJ 2 in human atherosclerotic lesions.In broad outline, atherosclerosis is considered to be a form of chronic inflammation.The early stage of atherosclerosis is characterized histopathologically by formation of fatty streaks composed of macrophage-derived foamy cells and exudate-rich extracellular matrix in the intima.The advanced stage of this disease is characterized by increased numbers of foamy macrophages and ulceration and calcification of the fibrously thickened intima.As shown in Fig.3, intense immunoreactivities for COX-2 (panel C) and 15d-PGJ 2 (panel D) were found to be localized in the cytoplasm of foamy macrophages identified in hematoxylin-eosin-stained (panel A) or CD68-immunostained (panel B) sections.Aortic wall areas showing atherosclerotic changes by guest on September 16, 2017 http://www.jbc.org/ Conversion of PGD 2 to 15d-PGJ 2 in cell-free medium and in phosphate-buffered saline.(A) Conversion of PGD 2 to 15d-PGJ 2 in cell-free medium.PGD 2 (1 mM) was OC. Human serum albumin was boiled for 5 min prior to incubation.