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• W2079644509 abstract "Prostaglandin D2(PGD2), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J2 series, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), 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 productionin vivo remain unclear. In the present study, we raised a monoclonal antibody specific to 15d-PGJ2 and determined its production in inflammation-related events. The monoclonal antibody (mAb11G2) was raised against the 15d-PGJ2-keyhole limpet hemocyanin conjugate and was found to recognize free 15d-PGJ2 specifically. The presence of 15d-PGJ2 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-PGJ2, suggesting that the PGD2 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 PGD2, which was associated with a significant increase in the extracellular 15d-PGJ2 levels, and the extracellular 15d-PGJ2production was reproduced by incubating PGD2 in a cell-free medium and in phosphate-buffered saline. Moreover, using a chiral high performance liquid chromatography method for separation of PGD2 metabolites, we established a novel metabolic pathway, in which PGD2 is converted to 15d-PGJ2 via an albumin-independent mechanism. Prostaglandin D2(PGD2), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J2 series, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), 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 productionin vivo remain unclear. In the present study, we raised a monoclonal antibody specific to 15d-PGJ2 and determined its production in inflammation-related events. The monoclonal antibody (mAb11G2) was raised against the 15d-PGJ2-keyhole limpet hemocyanin conjugate and was found to recognize free 15d-PGJ2 specifically. The presence of 15d-PGJ2 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-PGJ2, suggesting that the PGD2 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 PGD2, which was associated with a significant increase in the extracellular 15d-PGJ2 levels, and the extracellular 15d-PGJ2production was reproduced by incubating PGD2 in a cell-free medium and in phosphate-buffered saline. Moreover, using a chiral high performance liquid chromatography method for separation of PGD2 metabolites, we established a novel metabolic pathway, in which PGD2 is converted to 15d-PGJ2 via an albumin-independent mechanism. prostaglandins 15-deoxy-Δ12,14-PGJ2 prostaglandin D2 keyhole limpet hemocyanin 1-ethyl-3-(dimethylaminopropyl)carbodiimide sulfo-N-hydroxysuccinimide lipopolysaccharides cyclooxygense-2 enzyme-linked immunosorbent assay peroxisome proliferator-activated receptor γ Dulbecco's modified Eagle's medium fetal bovine serum high performance liquid chromatography bovine serum albumin phosphate-buffered saline liquid chromatography-mass spectrometry The prostaglandins (PGs)1 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 (1Smith W.L. Biochem. J. 1989; 259: 315-324Crossref PubMed Scopus (774) Google Scholar, 2Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar). 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 PGD2, PGE2, PGF2α, prostacyclin (PGI2), and thromboxane A2, through the action of specific PG synthetases. PGD2, 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 (3Giles H. Leff P. Prostaglandins. 1988; 35: 277-300Crossref PubMed Scopus (160) Google Scholar). The PGs are physiologically present in body fluids in picomolar-to-nanomolar concentrations (4Fukushima M. Eicosanoids. 1990; 3: 189-199PubMed Google Scholar); however, arachidonate metabolism is highly increased under several pathological conditions, including hyperthermia, infection, and inflammation (5Herschman H.R. Adv. Exp. Med. Biol. 1997; 407: 61-66Crossref PubMed Google Scholar), and local PG concentrations in the micromolar range have been detected at sites of acute inflammation (6Offenbacher S. Odle B.M. Van Dyke T.E. J. Periodontal Res. 1986; 21: 101-112Crossref PubMed Scopus (381) Google Scholar). In vitro, PGD2spontaneously converts into the cyclopentenone PGs of the J series, such as PGJ2, Δ12-PGJ2, and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) (see Fig. 1) (7Fitzpatrick F.A. Wynalda M.A. J. Biol. Chem. 1983; 258: 11713-11718Abstract Full Text PDF PubMed Google Scholar, 8Kikawa Y. Narumiya S. Fukushima M. Wakatsuka H. Hayaishi O. Proc. Natl. Acad. U. S. A. 1984; 81: 1317-1321Crossref PubMed Scopus (166) Google Scholar). It is not known whether this pathway is utilized in the organism, but it is clear that J2 prostanoids are synthesized in vivo. This is based on the observations that Δ12-PGJ2 is a natural component of human body fluids (9Hirata Y. Hayashi H. Ito S. Kikawa Y. Ishibashi M. Sudo M. Miyazaki H. Fukushima M. Narumiya S. Hayashi O. J. Biol. Chem. 1988; 263: 16619-16625Abstract Full Text PDF PubMed Google Scholar) and that Δ12-PGJ2 synthesis is suppressed by treatment with COX inhibitors (9Hirata Y. Hayashi H. Ito S. Kikawa Y. Ishibashi M. Sudo M. Miyazaki H. Fukushima M. Narumiya S. Hayashi O. J. Biol. Chem. 1988; 263: 16619-16625Abstract Full Text PDF PubMed Google Scholar). The natural precursor of PGJ2 derivatives appears to be PGD2, because its in vivo administration leads to a large increase in Δ12-PGJ2 (9Hirata Y. Hayashi H. Ito S. Kikawa Y. Ishibashi M. Sudo M. Miyazaki H. Fukushima M. Narumiya S. Hayashi O. J. Biol. Chem. 1988; 263: 16619-16625Abstract Full Text PDF PubMed Google Scholar). The cyclopentenone PGs have been reported to have their own unique spectrum of biological effects, including inhibition of macrophage-derived cytokine production (10Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. Nature. 1998; 391: 79-82Crossref PubMed Scopus (3260) Google Scholar, 11Jiang C. Ting A.T. Seed B. Nature. 1998; 391: 82-86Crossref PubMed Scopus (539) Google Scholar) and IκB kinase (12Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1203) Google Scholar,13Straus D. Pascual G. Li M. Welch J.S. Ricote M. Hsiang C.-H. Sengchanthalangsy L.L. Ghosh G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4844-4849Crossref PubMed Scopus (948) Google Scholar), induction of synoviocyte and endothelial cell apoptosis (14Bishop-Bailey D. Hla T. J. Biol. Chem. 1998; 274: 17042-17048Abstract Full Text Full Text PDF Scopus (407) Google Scholar), induction of glutathione S-transferase gene expression (15Kawamoto Y. Nakamura Y. Naito Y. Torii Y. Kumagai T. Osawa T. Ohigashi H. Satoh K. Imagawa M. Uchida K. J. Biol. Chem. 2000; 275: 11291-11299Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and intracellular oxidative stress (16Kondo M. Oya-Ito T. Kumagai T. Osawa T. Uchida K. J. Biol. Chem. 2001; 276: 12076-12083Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), and potentiation of apoptosis in activated macrophages (17Hortelano S. Castrillo A. Alvarez A.M. Bosca L. J. Immunol. 2000; 165: 6525-6531Crossref PubMed Scopus (114) Google Scholar). Furthermore, recent studies have reported that they can function as a feedback regulator of the inflammatory response (12Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1203) Google Scholar, 13Straus D. Pascual G. Li M. Welch J.S. Ricote M. Hsiang C.-H. Sengchanthalangsy L.L. Ghosh G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4844-4849Crossref PubMed Scopus (948) Google Scholar). 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 PGD2is the major prostaglandin in most tissues (4Fukushima M. Eicosanoids. 1990; 3: 189-199PubMed Google Scholar), it is likely that the cyclopentenone-type PGD2 metabolites are produced at a number of sites and may reach functionally significant levels in inflammation and its related disorders. In the present study, we raised a monoclonal antibody specific to 15d-PGJ2 and determined its endogenous production in human atherosclerotic lesions. Moreover, we investigated the intracellular and extracellular production of 15d-PGJ2 in the activated RAW264.7 macrophages in vitro and established a novel mechanism of transformation of PGD2 into the cyclopentenone-type PGJ2 derivatives, in which PGD2 is converted to 15d-PGJ2 via an albumin-independent mechanism. PGs were purchased from Cayman Chemical Co. (Ann Arbor, MI). Keyhole limpet hemocyanin (KLH) was obtained from Pierce. Horseradish peroxidase-linked anti-rabbit IgG immunoglobulin and ECL (enhanced chemiluminescence) Western blotting detection reagents were obtained from (Amersham Biosciences, Inc., Buckinghamshire, UK). Lipopolysaccharides (LPS) were obtained from Sigma Chemical Co. (St Louis, MO). Murine RAW264.7 macrophages were kind gifts from Dr. A. Murakami (Kinki University) and Dr. W. Maruyama (National Institutes of Longevity Sciences), respectively. RAW264.7 cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml), and 0.2% NaHCO3, at 37 °C in an atmosphere of 95% air and 5% CO2. Cells post-confluency were exposed to LPS (10 μg/ml) in DMEM containing 10% FBS. 15d-PGJ2 was coupled to KLH by 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (sulfo-NHS) as described by Grabarek and Gergely (20Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (726) Google Scholar). Female BALB/c mice were immunized three times with the 15d-PGJ2-KLH conjugate. Spleen cells from the immunized mice were fused with P3 murine myeloma cells and cultured in hypoxanthine/aminopterin/thymidine selection medium. Culture supernatants of the hybridoma were screened using ELISA, employing pairs of wells of microtiter plates on which was absorbed 15d-PGJ2-BSA conjugate as the antigen (0.5 μg of protein/well). After incubation with 100 μl of hybridoma supernatants, and with intervening washes with PBS containing 0.05% Tween 20 (PBS/Tween), the wells were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG, followed by a substrate solution containing 1 mg/ml p-nitrophenyl phosphate. Hybridoma cells corresponding to supernatants that were positive on 15d-PGJ2-BSA were then cloned by limiting dilution. After repeated screening, two clones were obtained. Among them, clone 11G2 showed the most distinctive recognition of 15d-PGJ2-BSA. A gel was transblotted onto a nitrocellulose membrane, incubated with Block Ace (40 mg/ml) for blocking, washed, and treated with anti-COX-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). This procedure was followed by the addition of horseradish peroxidase conjugated to a goat anti-mouse IgG F(ab′)2 fragment and ECL reagents (Amersham Biosciences, Inc.). The bands were visualized by exposure of the membranes to autoradiography film. Cells were fixed overnight in PBS containing 2% paraformaldehyde and 0.2% picric acid at 4 °C. Membranes were permeabilized by exposing the fixed cells to PBS containing 0.3% Triton X-100. The cells were then incubated sequentially in PBS solutions containing 2% BSA and primary antibody (mAb11G2). The cells were then incubated for 1 h in the presence of fluorescein isothiocyanate-labeled anti-rabbit IgG (Dako) or Cy3-labeled goat anti-mouse IgG (Amersham Biosciences, Inc.), rinsed with PBS containing 0.3% Triton X-100, and covered with anti-fade solution. Images of cellular immunofluorescence were acquired using a confocal laser scanning microscope (Fluoroview, Olympus Optical Co., Ltd, Tokyo) with a ×40 objective (488-nm excitation and 518-nm emission). Extracellular PGD2 and 15d-PGJ2 levels were determined in the culture medium of RAW264.7 macrophages activated with LPS. At the end of the incubation period, the medium was collected and stored at −80 °C. For determination of PGD2, a solid-phase enzyme immunoassay (Cayman Chemical) was performed as suggested by the manufacturer, and the PGD2 level was determined using a standard curve and a linear log-logit transformation. For determination of 15d-PGJ2 in the cell culture medium, the medium (5 ml) was extracted immediately with 3 × 5 ml of ethyl acetate, and the solvent was evaporated under nitrogen. The residue was reconstituted in 200 μl of PBS containing 0.05% Tween 20 (PBS/Tween) and incubated with the antibody (mAb11G2) for 20 h at 4 °C to yield competitor/antibody mixtures containing antibody at 0.2 μg/ml. A 100-μl aliquot of the competitor/antibody mixture was added to each well and incubated for 2 h at 37 °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-PGJ2 levels in the medium were determined using a standard curve and a linear log-logit transformation. For determination of 15d-PGJ2upon in vitro incubation of PGD2 in cell-free medium or PBS, an aliquot (200 μl) of the reaction mixtures was directly subjected to the competitive ELISA assay. 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 (Sakura Fine Technical Co., Tokyo, Japan), stored at −80 °C, and cut into 6-m-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) 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. Incubation of PGs was carried out in PBS at 37 °C. Samples were withdrawn periodically and extracted immediately with 3 × 0.2 ml of ethyl acetate. The solvent was evaporated under nitrogen, and the residue was reconstituted in 50 μl of the chromatographic mobile phase. PG metabolites were separated on a ChiralPak AD-RH column (0.46 × 15 cm, Daicel Chemical Industries, Ltd., Osaka, Japan) eluted with a linear gradient of acetonitrile/water/acetic acid (90/10/0.01, v/v) (solvent A) − acetonitrile (solvent B) (time = 0–5 min, 100% A; 60 min, 0% A), at a flow rate of 0.8 ml/min. The elution profiles were monitored by UV absorbance at 200–400 nm. Liquid chromatography-mass spectrometry (LC-MS) was measured with a Jasco PlatformII-LC instrument. 15d-PGJ2 is believed to be physiologically formed through the nonenzymatic conversion of PGD2 (Fig. 1) but has never been definitively proven to exist in vivo. In view of its biological significance, it is critical to provide evidence that 15d-PGJ2 is endogenously produced in vivo. To this end, we produced a monoclonal antibody directed to 15d-PGJ2. The antibody was raised against the 15d-PGJ2-KLH conjugate, which was prepared from the reaction of KLH with 15d-PGJ2 in the presence of EDC and sulfo-NHS (Fig. 2 A). During the preparation of the monoclonal antibodies, hybridomas were selected on the basis of the ability of their antibodies to bind to the 15d-PGJ2-BSA conjugate. After repeated screening, three clones were obtained. Among them, the antibody produced by the clone 11G2 most strongly recognized the 15d-PGJ2-BSA conjugate but did not recognize the native BSA (Fig. 2 B). The antibody was then tested for immunoreactivity with PGs. As shown in Fig.2 C, mAb11G2 recognized 15d-PGJ2 most significantly. Both PGJ2 and Δ12-PGJ2 also served as weak antigens, although their inhibitions were about 50% times lower than 15d-PGJ2. The antibody did not cross-react with other PGs, such as PGA2, PGB2, PGD2, PGE2, PGF2α, and PGI2 (data not shown). Taken together, these data indicated that mAb11G2 was directed almost exclusively against the structure of 15d-PGJ2. We first examined the in vivopresence of 15d-PGJ2 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-PGJ2 (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 displayed no significant 15-PGJ2immunoreactivity. 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-PGJ2 in vivo. To examine whether 15d-PGJ2 is produced in response to inflammatory stimuli in vitro, RAW264.7 macrophages were exposed to a pro-inflammatory agent (LPS) and 15d-PGJ2 produced within the cells was stained with mAb11G2. As shown in Fig. 4 Aand 4 B (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-PGJ2 immunoreactivity in essentially all cells (Fig. 4 B, panels c and d). An immunofluorescence double labeling of the activated cells revealed almost identical cellular distribution of COX-2 and 15d-PGJ2 (Fig. 4 C). These results were consistent with the in vivo observations that 15d-PGJ2 was detected intracellularly in the macrophage-derived foam cells in atherosclerotic lesions (Fig. 3). Taking these in vivo and in vitro data together, it is evident that the production of 15d-PGJ2 is highly accelerated in the cells during inflammatory processes. 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. Hortelanoet al. (17Hortelano S. Castrillo A. Alvarez A.M. Bosca L. J. Immunol. 2000; 165: 6525-6531Crossref PubMed Scopus (114) Google Scholar) have also suggested that 15d-PGJ2may contribute to the resolution of inflammation as a paracrine factor. The extracellular production of 15d-PGJ2 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-PGJ2 is extracellularly produced during inflammation, the levels of 15d-PGJ2 in the culture medium of the activated macrophages were measured by a competitive ELISA assay. As shown in Fig.5 A, the calibration range (0.1–10 nmol) of the standard curve for 15d-PGJ2 was obtained. In parallel with the COX-2 up-regulation, the extracellular levels of PGD2 in the LPS-stimulated RAW264.7 macrophages were significantly increased (Fig. 5 B). In addition, accompanied by the production of PGD2, a significant amount of 15d-PGJ2 was accumulated in the culture medium (Fig.5 C). The levels of these PGs reached maximums after 12 h of incubation and then decreased thereafter. These data proved that 15d-PGJ2 was produced extracellularly during inflammatory processes. Because PGD2 is known to be converted sequentially to the J2 derivatives of PGs in vitro (Fig. 1) (21Fukushima M. Prostaglandins Leukot. Essent. Fatty Acids. 1992; 47: 1-12Abstract Full Text PDF PubMed Scopus (187) Google Scholar), it was anticipated that 15d-PGJ2could be produced extracellularly in the medium through the metabolism of PGD2. To examine the extracellular conversion of PGD2 to 15d-PGJ2, 1 mmPGD2 was incubated in the cell-free medium and the formation of 15d-PGJ2 was examined. As shown in Fig.6 A, 15d-PGJ2 was detectable by the ELISA assay within 1 h after initiating the incubation. The concentration of 15d-PGJ2 increased almost linearly from 0 to 8 h and reached a maximum concentration at 24 h. The amount corresponded to the 8% PGD2 that disappeared during incubation. The maximum levels were maintained for the duration of the experiment. Serum albumin has been identified as the plasma protein that can catalyze the in vitro transformation of PGD2into the J2 derivatives of PGs in aqueous buffer (7Fitzpatrick F.A. Wynalda M.A. J. Biol. Chem. 1983; 258: 11713-11718Abstract Full Text PDF PubMed Google Scholar). Accordingly, to examine whether serum albumin contained in the medium functioned as a catalyst in the conversion of PGD2 to 15d-PGJ2, 1 mm PGD2 was incubated in PBS containing 10 mg/ml human serum albumin. As expected, PGD2 was similarly converted to 15d-PGJ2 in PBS containing serum albumin (Fig. 6 B). 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-PGJ2 from PGD2. The data (Fig. 6 B) contradict the mechanism of PGD2metabolism (Fig. 1), in which PGD2 is sequentially converted to PGJ2, Δ12-PGJ2, and 15d-PGJ2 and the route of conversion leading from PGJ2 to Δ12-PGJ2 is catalyzed by serum albumin, therefore, suggesting the presence of an albumin-independent mechanism for production of 15d-PGJ2. To establish a mechanism of transformation of PGD2 into the cyclopentenone-type PGJ2 derivatives, we developed a chiral HPLC method for separation of PGD2 metabolites and investigated the conventional PGD2 metabolic pathway in detail. When PGD2 (1 mm) was incubated in PBS for 24 h, three products (a, b, andc) were mainly detected (Fig.7 A, peaks a,b, and c, respectively). 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 cochromatography with authentic PGs, the products a,b, and c were suggested to be PGJ2, 15d-PGD2, and 15d-PGJ2, respectively. The identification of the products was finally done by LC-MS analysis of the products, which showed molecular ion peaks atm/z 316.4 (M − H2O)+ (a), 334.6 (M)+(b), and 316.9 (M)+ (c). It is striking to note that Δ12-PGJ2, which was reported to be an immediate metabolite of PGJ2 in the transformation of PGD2 (21Fukushima M. Prostaglandins Leukot. Essent. Fatty Acids. 1992; 47: 1-12Abstract Full Text PDF PubMed Scopus (187) Google Scholar), was not detected in the incubation of PGD2 alone in PBS. Both PGJ2 and 15d-PGD2 were estimated to be generated through dehydration reactions at C9 and C15 of PGD2, respectively, and to be further dehydrated to 15d-PGJ2. Hence, to identify the direct precursor of 15d-PGJ2, PGJ2 and 15d-PGD2 were individually incubated in PBS at 37 °C and the products were analyzed by chiral-phase HPLC. As shown in Fig. 7 (B andC), PGJ2 was stoichiometrically converted to 15d-PGJ2, whereas no further conversion of 15d-PGD2 was observed (data not shown). In addition, we were unable to detect Δ12-PGJ2 upon incubation of PGJ2 and 15d-PGD2, suggesting that Δ12-PGJ2 was not involved in the spontaneous conversion of PGD2 to the PGJ2derivatives. These data indicate that the PGD2 is primarily converted to three products, including PGJ2, 15d-PGD2, and 15d-PGJ2, among which PGJ2 is a direct precursor of 15d-PGJ2. Serum albumin has been previously identified as the endogenous catalyst of PGD2 metabolism (7Fitzpatrick F.A. Wynalda M.A. J. Biol. Chem. 1983; 258: 11713-11718Abstract Full Text PDF PubMed Google Scholar, 8Kikawa Y. Narumiya S. Fukushima M. Wakatsuka H. Hayaishi O. Proc. Natl. Acad. U. S. A. 1984; 81: 1317-1321Crossref PubMed Scopus (166) Google Scholar, 9Hirata Y. Hayashi H. Ito S. Kikawa Y. Ishibashi M. Sudo M. Miyazaki H. Fukushima M. Narumiya S. Hayashi O. J. Biol. Chem. 1988; 263: 16619-16625Abstract Full Text PDF PubMed Google Scholar). In addition, these workers have identified Δ12-PGJ2 as the major product in the albumin-catalyzed PGD2 metabolism. Hence, to characterize the albumin-dependent PGD2metabolic pathway, PGD2 was incubated in PBS containing human serum albumin (10 mg/ml) at 37 °C, and the products were analyzed by chiral-phase HPLC. As shown in Fig.8 A (upper chromatogram), incubation of PGD2 with human serum albumin yielded, not only the same products (15d-PGD2, PGJ2, and 15d-PGJ2) as those detected in the spontaneous conversion of PGD2 in albumin-free solution (Fig. 7 A) but also a major product which cochromatographed on HPLC with Δ12-PGJ2. The LC-MS analysis of the product showed molecular ion (M)+ peak atm/z 334.8, which coincided with the theoretical molecular weight of Δ12-PGJ2. Thus, the product was determined to be Δ12-PGJ2. It was observed that PGJ2 was converted to both 15d-PGJ2 and Δ12-PGJ2 in the presence of human serum albumin (Fig. 8 A,middle). Moreover, no further conversion of Δ12-PGJ2 was observed (Fig. 8 A,bottom), indicating that Δ12-PGJ2represents one of the terminal product of PGD2 metabolism. Isomerization of PGJ2 to Δ12-PGJ2was dependent on the incubation time and the concentration of human serum albumin: When human serum albumin was present in excess, PGJ2 was almost stoichiometrically converted to Δ12-PGJ2 (Fig. 8 B). Boiling of serum albumin markedly decreased the formation of Δ12-PGJ2 from PGJ2 but did not affect the formation of 15d-PGJ2 (Fig. 8 C), suggesting that Δ12-PGJ2 is formed by the enzymatic action of albumin while 15d-PGJ2 is a nonenzymatically formed product. Taken together, these data suggest that PGD2 is sequentially converted to PGJ2 and 15d-PGJ2 in an albumin-independent manner and serum albumin is involved only in the process leading from PGJ2 to Δ12-PGJ2 (Fig.9). 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-PGJ2-protein conjugate from the native protein. Characterization of the antibody revealed that the monoclonal antibody was directed almost exclusively against free 15d-PGJ2 (Fig. 2). It was expected that mAb11G2 would be useful in assessing the endogenous production of 15d-PGJ2in response to inflammatory stimuli. In vivo detection of 15d-PGJ2 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. 3 C). In addition, 15d-PGJ2 was also found to localize predominantly with the lesional macrophages (Fig. 3 D). These observations raise the possibility that 15d-PGJ2 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 stimulat" @default.
• W2079644509 created "2016-06-24" @default.
• W2079644509 creator A5004162735 @default.
• W2079644509 creator A5005312770 @default.
• W2079644509 creator A5035336165 @default.
• W2079644509 creator A5055822025 @default.
• W2079644509 creator A5070408640 @default.
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• W2079644509 date "2002-03-01" @default.
• W2079644509 modified "2023-10-16" @default.
• W2079644509 title "15-Deoxy-Δ12,14-prostaglandin J2" @default.
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