Delta 12-prostaglandin J2, a plasma metabolite of prostaglandin D2, causes eosinophil mobilization from the bone marrow and primes eosinophils for chemotaxis

PGD(2), a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD(2) is rapidly transformed into its major metabolite delta(12)-PGJ(2), the effect of which on eosinophil migration has not yet been characterized. In this study we found that delta(12)-PGJ(2) was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD(2), PGJ(2), or 15-deoxy-delta(12,14)-PGJ(2). Moreover, pretreatment of eosinophils with delta(12)-PGJ(2) markedly enhanced the chemotactic response to eotaxin, and in this respect delta(12)-PGJ(2) was more effective than PGD(2). delta(12)-PGJ(2)-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that delta(12)-PGJ(2) signaled through the same receptor, CRTH2, as PGD(2). Finally, delta(12)-PGJ(2) was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that delta(12)-PGJ(2) is present in the systemic circulation at relevant levels, a role for this PGD(2) metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.     

Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.     

The role of the prostaglandin D2 receptor, DP, in eosinophil trafficking

Prostaglandin (PG) D2 is a major mast cell product that acts via two receptors, the D-type prostanoid (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors. Whereas CRTH2 mediates the chemotaxis of eosinophils, basophils, and Th2 lymphocytes, the role of DP has remained unclear. We report in this study that, in addition to CRTH2, the DP receptor plays an important role in eosinophil trafficking. First, we investigated the release of eosinophils from bone marrow using the in situ perfused guinea pig hind limb preparation. PGD2 induced the rapid release of eosinophils from bone marrow and this effect was inhibited by either the DP receptor antagonist BWA868c or the CRTH2 receptor antagonist ramatroban. In contrast, BWA868c did not inhibit the release of bone marrow eosinophils when this was induced by the CRTH2-selective agonist 13,14-dihydro-15-keto-PGD2. In additional experiments, we isolated bone marrow eosinophils from the femoral cavity and found that these cells migrated toward PGD2. We also observed that BWA868c inhibited this response to a similar extent as ramatroban. Finally, using immunohistochemistry we could demonstrate that eosinophils in human bone marrow specimens expressed DP and CRTH2 receptors at similar levels. Eosinophils isolated from human peripheral blood likewise expressed DP receptor protein but at lower levels than CRTH2. In agreement with this, the chemotaxis of human peripheral blood eosinophils was inhibited both by BWA868c and ramatroban. These findings suggest that DP receptors comediate with CRTH2 the mobilization of eosinophils from bone marrow and their chemotaxis, which might provide the rationale for DP antagonists in the treatment of allergic disease.     

9alpha,11beta-PGF2 and its stereoisomer PGF2alpha are novel agonists of the chemoattractant receptor, CRTH2

CRTH2 is a recently described chemoattractant receptor for the prostaglandin, PGD(2), expressed by Th2 cells, eosinophils and basophils, and believed to play a role in allergic inflammation. Here we describe the potency of several PGD(2) metabolites at the receptor to induce cell migration and activation. We report for the first time that the PGD(2) metabolite, 9alpha,11beta-PGF(2), and its stereoisomer, PGF(2alpha), are CRTH2 agonists. 9alpha,11beta-PGF(2) is a major metabolite produced in vivo following allergen challenge, whilst PGF(2alpha) is generated independently of PGD synthetase, with implications for CRTH2 signalling in the presence or absence of PGD(2) production.     

11-Dehydro-thromboxane B2, a stable thromboxane metabolite, is a full agonist of chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) in human eosinophils and basophils

Thromboxane (TX) A(2), a cyclooxygenase-derived mediator involved in allergic responses, is rapidly converted in vivo to a stable metabolite, 11-dehydro-TXB(2), which is considered to be biologically inactive. In this study, we found that 11-dehydro-TXB(2), but not the TXA(2) analogue U46,619 or TXB(2), activated eosinophils and basophils, as assayed by flow cytometric shape change. 11-Dehydro-TXB(2) was also chemotactic for eosinophils but did not induce, nor inhibit, platelet aggregation. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is an important chemoattractant receptor expressed by eosinophils, basophils, and TH2 lymphocytes, and prostaglandin (PG)D(2) has been shown to be its principal ligand. 11-Dehydro-TXB(2) induced calcium flux mainly from intracellular stores in eosinophils, and this response was desensitized after stimulation with PGD(2) but not other eosinophil chemoattractants. Shape change responses of eosinophils and basophils to 11-dehydro-TXB(2) were inhibited by the thromboxane (TP)/CRTH2 receptor antagonist ramatroban, but not the selective TP antagonist SQ29,548, and were insensitive to pertussis toxin. The phospholipase C inhibitor U73,122 attenuated both 11-dehydro-TXB(2)- and PGD(2)-induced shape change. 11-Dehydro-TXB(2) also induced the chemotaxis of BaF/3 cells transfected with hCRTH2 but not naive BaF/3 cells. At a threshold concentration, 11-dehydro-TXB(2) had no antagonistic effect on CRTH2-mediated responses as induced by PGD2. These data show that 11-dehydro-TXB(2) is a full agonist of the CRTH2 receptor and hence might cause CRTH2 activation in cellular contexts where PGD-synthase is not present. Given its production in the allergic lung, antagonism of the 11-dehydro-TXB(2)/CRTH2axis may be of therapeutic relevance.     

Prostaglandin D2 affects the maturation of human monocyte-derived dendritic cells: consequence on the polarization of naive Th cells

Among the factors produced at inflammatory sites and those capable of modulating dendritic cell (DC) functions, PGD(2) may be important in the outcome of immune responses. The biological roles for PGD(2) are in part effected through two plasma membrane G protein-coupled receptors: the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes (CRTH2). In this report, we studied the effects of PGD(2) and of its major physiological metabolite, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), on the functions of human monocyte-derived DC. First, we show that PGD(2) exerts in vitro chemotactic effects on monocytes via CRTH2 activation while it inhibits the chemokine-driven migration of monocyte-derived DC through DP. We also report that PGD(2) and 15d-PGJ(2) alter the LPS- and allergen-induced DC maturation and enhance the CD80/CD86 ratio on mature DC in a DP- and CRTH2-independent manner. Moreover, PGD(2) and 15d-PGJ(2) strongly reduce the secretion of the Th1 promoting cytokine IL-12 and affect the synthesis of chemokines involved in Th1 cell chemotaxis, particularly CXCL10. Inhibition of cytokine/chemokine secretion implicates at least in part DP, but not CRTH2. The effects exerted by PGD(2) are associated with the phosphorylation of CREB, but do not parallel with the deactivation of the NF-kappa B and mitogen-activated protein kinase pathways. In contrast, 15d-PGJ(2) seems to target other cellular proteins. Finally, in a model of Th CD45RA(+) differentiation induced by allergen- and superantigen-pulsed DC, PGD(2) impacts on the orientation of the immune response by favoring a Th2 response.     

Cutting edge: chemoattractant receptor-homologous molecule expressed on Th2 cells plays a restricting role on IL-5 production and eosinophil recruitment

PGs play key regulatory roles in inflammation and immunity. PGD2, released from mast cells and Th2 cells during allergic responses, has recently been shown to target a novel receptor, chemoattractant receptor-homologous molecule expressed TH2 cells (CRTH2), in addition to the classic PGD (DP) receptor. CRTH2 is expressed on Th2 cells and eosinophils and mediates chemotaxis of these cells to PGD2. Thus, CRTH2 is thought to be a key receptor mediating eosinophil and Th2 cell recruitment during allergic responses. To examine the role of CRTH2 in this context in vivo, we generated CRTH2 knockout mice. Surprisingly, in an allergic inflammatory model of asthma, CRTH2 knockout mice showed enhanced eosinophil recruitment into the lung compared with wild-type littermate mice. This is consistent with our observation that CRTH2 knockout cells produce significantly higher amounts of IL-5 and IL-3 in vitro. These results suggest a nonredundant role of CRTH2 in restricting eosinophilia and allergic response in vivo.     

The second PGD(2) receptor CRTH2: structure,properties, and functions in leukocytes

Prostaglandin (PG) D(2) plays a broad range of physiological and pathophysiological functions. Until just a few years ago, it was thought that most of the biological actions of PGD(2) are mediated via the classical PGD(2) receptor DP. Recently, we identified a second PGD(2) receptor, chemoattractant receptor-homologous molecule expressed on T helper (Th)2 cells (CRTH2), with different functions relative to DP. Here, we review the recent findings on the structure, tissue distribution, ligand selectivity, signalling pathways, and functions in leukocytes of this receptor. The data suggest that the PGD(2)/CRTH2 system play important roles in allergic inflammation through its stimulatory effects on Th2 cells, eosinophils, and basophils.     

Cycloheximide reduces PGD2 or delta 12-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD2 or delta 12-PGJ2 (a biologically active metabolite of PGD2), we examined the effect of various compounds on PGD2 or delta 12-PGJ2 cytotoxicity, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD2 cytotoxicity on NCG cells. When delta 12-PGJ2 was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and alpha-amanitin did not affect PGD2 or delta 12-PGJ2 cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD2 or delta 12-PGJ2 exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD2 or delta 12-PGJ2.     

Gene structure and functional properties of mouse CRTH2, a prostaglandin D2 receptor

CRTH2, the second receptor for prostaglandin D(2) (PGD(2)), is thought to play a role in allergic inflammations through the induction of chemotactic migration and/or the activation of Th2, eosinophils, and basophils, in humans. We previously identified the mouse CRTH2 homolog of human CRTH2 and suggest that animal models would provide a clear understanding on the precise function of CRTH2 in allergic disorders. To this end we have confirmed that mouse CRTH2 is similar in gene structure to human CRTH2 and revealed that mouse CRTH2 is predominantly expressed in the eosinophils derived from IL-5-transgenic mice. Moreover, mouse CRTH2 harbors the ability to bind PGD(2) with high affinity and intracellular Ca(2+) mobilization in a Gi-dependent manner and chemotactic responses in several transfected cell lines. The results demonstrated here indicate that mouse CRTH2 is the functional ortholog of human CRTH2 and paves the way for future analysis of the in vivo functions of CRTH2.     

Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor

PGD(2) plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35-55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD(2)-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.     

15-Deoxy-delta 12,14-prostaglandins D2 and J2 are potent activators of human eosinophils

15-Deoxy-Delta(12,14)-PDJ(2) (15d-PGJ(2)) is a degradation product of PGD(2) that has been proposed as an anti-inflammatory compound because of its various inhibitory effects, some of which are mediated by peroxisome proliferator-activated receptor-gamma. In contrast to its reported inhibitory effects on macrophages and other cells, we found that this compound is a potent activator of eosinophils, inducing calcium mobilization, actin polymerization, and CD11b expression. It is selective for eosinophils, having little or no effect on neutrophils or monocytes. 15d-PGJ(2) has an EC(50) of approximately 10 nM, similar to that of its precursor, PGD(2). The concentrations of 15d-PGJ(2) required to activate eosinophils are thus much lower than those required for its anti-inflammatory effects (usually micromolar). 15-Deoxy-Delta(12,14)-prostaglandin D(2) (15d-PGD(2)) is also a potent activator of eosinophils, with an EC(50) about the same as that of PGD(2), whereas Delta(12)-PGJ(2) is slightly less potent. Eosinophils pretreated with PGD(2) no longer respond to 15d-PGJ(2), and vice versa, but in both cases the cells still respond to another eicosanoid proinflammatory mediator, 5-oxo-6,8,11,14-eicosatetraenoic acid. This indicates that the effects of 15d-PGJ(2) are mediated by the DP(2)/chemoattractant receptor-homologous molecule expressed on Th2 cells that has recently been identified in eosinophils. 15d-PGJ(2) is selective for the DP(2) receptor, in that it has no effect on DP(1) receptor-mediated adenylyl cyclase activity in platelets. We conclude that 15d-PGJ(2) and 15d-PGD(2) are selective DP(2) receptor agonists that activate human eosinophils with potencies at least 100 times greater than those for the proposed anti-inflammatory effects of 15d-PGJ(2) on other cells.     

Cutting edge: agonistic effect of indomethacin on a prostaglandin D2 receptor, CRTH2.

Indomethacin is a widely used nonsteroidal anti-inflammatory drug and is generally known to exhibit its multiple biological functions by inhibiting cyclooxygenases or activating peroxisome proliferator-activated receptors. In this study, we present evidence demonstrating that the novel PGD(2) receptor chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is another functional target for indomethacin. Indomethacin induced Ca(2+) mobilization in CRTH2-transfected K562 cells at submicromolar concentrations (approximate EC(50), 50 nM) in a G(alphai)-dependent manner as PGD(2) did. Other nonsteroidal anti-inflammatory drugs (aspirin, sulindac, diclofenac, and acemetacin) had no such effect even at micromolar concentrations. In chemotaxis assay, three CRTH2-expressing cell types, Th2 cells, eosinophils, and basophils, were all significantly attracted by indomethacin (EC(50), 50-500 nM) as well as by PGD(2) (EC(50), 2-20 nM), and the effects of indomethacin were blocked by anti-CRTH2 mAb. These results suggest the involvement of CRTH2 in mediating some of therapeutic and/or unwanted side effects of indomethacin, independently of cyclooxygenases and peroxisome proliferator-activated receptors.     

Prostaglandin D2 and its metabolites induce caspase-dependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism

Many inflammatory mediators retard granulocyte apoptosis. Most natural PGs studied herein (e.g., PGE(2), PGA(2), PGA(1), PGF(2 alpha)) either delayed apoptosis or had no effect, whereas PGD(2) and its metabolite PGJ(2) selectively induced eosinophil, but not neutrophil apoptosis. This novel proapoptotic effect does not appear to be mediated via classical PG receptor ligation or by elevation of intracellular cAMP or Ca(2+). Intriguingly, the sequential metabolites Delta(12)PGJ(2) and 15-deoxy-Delta(12,) Delta(14)-PGJ(2) (15dPGJ(2)) induced caspase-dependent apoptosis in both granulocytes, an effect that did not involve de novo protein synthesis. Despite the fact that Delta(12)PGJ(2) and 15dPGJ(2) are peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators, apoptosis was not mimicked by synthetic PPAR-gamma and PPAR-alpha ligands or blocked by an irreversible PPAR-gamma antagonist. Furthermore, Delta(12)PGJ(2) and 15dPGJ(2) inhibited LPS-induced I kappa B alpha degradation and subsequent inhibition of neutrophil apoptosis, suggesting that apoptosis is mediated via PPAR-gamma-independent inhibition of NF-kappa B activation. In addition, we show that TNF-alpha-mediated loss of cytoplasmic I kappa B alpha in eosinophils is inhibited by 15dPGJ(2) in a concentration-dependent manner. The selective induction of eosinophil apoptosis by PGD(2) and PGJ(2) may help define novel therapeutic pathways in diseases in which it would be desirable to specifically remove eosinophils but retain neutrophils for antibacterial host defense. The powerful proapoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2) in both granulocyte types suggest that these natural products control the longevity of key inflammatory cells and may be relevant to understanding the control and resolution of inflammation.     

Prostaglandin D2 receptors DP and CRTH2 in the pathogenesis of asthma

Prostaglandin D2 (PGD2) is a major prostanoid produced mainly by mast cells in allergic diseases, including bronchial asthma. However, its role in the pathogenesis of asthma remains unclear. PGD2-induced vasodilatation and increased permeability are well-known classical effects that may facilitate transendothelial migration of inflammatory cells, such as eosinophils, mast cells, lymphocytes, and monocytes in allergic inflammation. These effects are initiated via a PGD2 receptor, D prostanoid receptor (DP), and are referred to as DP-mediated vasodilation-extravasation. Recently, novel functions of DP have been identified. Furthermore, a novel and different receptor of PGD2, CRTH2, has been discovered. To date, DP and CRTH2 have been shown to be major PGD(2)-related receptors that have pivotal roles in mediating allergic diseases by effects such as directly regulating the migration of inflammatory cells and controlling the production of cytokines and lipid mediators. Available evidence suggests that CRTH2 and DP may collaborate in allergic inflammation. This review focuses on the novel roles of DP and CRTH2 in the initiation and maintenance of allergy.     

A novel PGD(2) receptor expressed in eosinophils

PGD(2) is a major product of arachidonic acid metabolism by mast cells and is released in the lungs following allergen challenge. Activation of the classic PGD(2) receptor (DP receptor) results in stimulation of adenylyl cyclase, resulting in inhibition of platelet aggregation and smooth muscle relaxation. A second PGD(2) receptor has recently been identified and designated as the DP(2) receptor, or chemoattractant receptor-homologous molecule expressed on Th2 cells. PGD(2) acts through the DP(2) receptor to induce eosinophil chemotaxis, actin polymerization, calcium mobilization, and adhesion molecule expression. The most potent DP(2) receptor agonist yet identified is 15R-methyl-PGD(2), which has the unnatural R configuration at C(15). 15-Deoxy-Delta(12,14)-PGJ(2) is also a potent DP(2) receptor agonist that activates eosinophils at concentrations much lower than those required for its anti-inflammatory effects. Because of its critical location in the lung and its potent effects on eosinophils, PGD(2) may be an important proinflammatory mediator in asthma.     

Occurrence of 9-deoxy-delta 9,delta 12-13,14-dihydroprostaglandin D2 in human urine

We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.     

Stimulation of NGF expression and secretion in 3T3-L1 adipocytes by prostaglandins PGD2, PGJ2, and Delta12-PGJ2

Nerve growth factor (NGF) has recently been shown to be secreted from white adipocytes, its production being strongly stimulated by the proinflammatory cytokine tumor necrosis factor-alpha. In this study, we have examined whether a series of prostaglandins and other inflammation-related factors also stimulate NGF expression and secretion by adipocytes, using 3T3-L1 cells. Although interleukin (IL)-1beta, IL-10, and IL-18 each induced a small decrease in NGF mRNA level in 3T3-L1 adipocytes, there was no significant effect of these cytokines on NGF secretion. A small reduction in NGF expression and/or secretion was also observed with adiponectin and prostaglandins PGE(2), PGF(2alpha), and PGI(2). In marked contrast, prostaglandin PGD(2) induced a major, dose-dependent increase (up to 20- to 40-fold) in NGF expression and secretion. The PGD(2) metabolites, PGJ(2) and Delta(12)-PGJ(2), also induced major increases (up to 30-fold) in NGF production. A further metabolite of PGJ(2), 15-deoxy-Delta(12,14)-PGJ(2), a peroxisome proliferator-activated receptor-gamma agonist, led paradoxically to a small increase in NGF mRNA level but a fall in NGF secretion. Both PGD(2) and PGJ(2) induced significant increases in NGF gene expression by 4 h after their addition. It is concluded that PGD(2) and the J series prostaglandins, PGJ(2) and Delta(12)-PGJ(2), can play a significant role in the regulation of NGF production by white adipocytes. These results provide support for the view that NGF is an important inflammatory response protein, as well as a target-derived neurotrophin, in white adipose tissue.     

delta 12-Prostaglandin J2, an ultimate metabolite of prostaglandin D2 exerting cell growth inhibition

L-1210 murine leukemia cells were exposed to prostaglandin D2 (PGD2), 10 micrograms/ml, in culture medium for various time, and subsequent cell growth was observed. More than 24 h exposure to PGD2 was required to inhibit cell growth almost completely. During this period, PGD2 degraded time-dependently into several products. The major product was identified as delta 12-PGJ2 by TLC, UV and mass spectra. When delta 12-PGJ2 was added to cells instead of PGD2, it evoked growth inhibition with much shorter contact time than PGD2. In addition, when the medium containing PGD2 was preincubated at 37 degrees C for 24 h, it elicited growth inhibition with only 6 h contact with cells. Furthermore, when the medium containing PGD2 was changed every 6 h during 24 h exposure time to cells, no significant growth inhibition was observed. These results suggested that PGD2 per se has little, if any, growth inhibitory activity, and delta 12-PGJ2 is an ultimate metabolite exerting growth inhibition. This action appears to be independent of cAMP, since delta 12-PGJ2 was virtually inactive in raising intracellular cAMP levels.