Prostaglandin D2 in cultured capillary and microvascular endothelium of human brain.

Production of prostaglandin D2 (PGD2) was investigated in cultured endothelial cells derived from capillaries and microvessels (small and large) of human brain using radioimmunoassays. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester and calcium ionophore greatly stimulated the secretion of endothelial PGD2. Secretion of PGD2 induced by vasoconstricting peptides, angiotensin II and arginine-vasopressin, was almost completely abolished by their respective specific receptor antagonists [Sar1, Ala8]-Ang II and [1-6(beta-mercapto-beta,beta-cyclopentamethylene propionic acid) 2-O-methyltyrosine]. Thus, the augmented production of PGD2 by angiotensin II and arginine-vasopressin is a receptor-mediated event. It also indicates that the EC have specific angiotensin II and arginine-vasopressin (V1) receptors. This study represents the first demonstration of vasoactive agents modulating PGD2 production in capillary and microvascular endothelium of human brain.     

Changes in the plasma prostaglandin F2 alpha metabolite before and during spontaneous labor and labor induced by amniotomy, oxytocin and prostaglandin E2

To elucidate the role of endogenous prostaglandin F2 alpha in spontaneous and induced labor, plasma concentrations of 13, 14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) were determined before the onset of labor, at onset of labor, during active labor, at the crowning of the fetal head, and 1 and 2 hours after delivery. Patients in spontaneous labor and labor induced by amniotomy, oxytocin, and prostaglandin E2 were studied. The levels of plasma PGFM in patients who entered spontaneous labor fell 2 to 3 weeks before delivery, whereas those in the induced labor group did not change until the time of induction. Although the levels of PGFM rose gradually with the progress of labor in all cases, the levels in the spontaneous labor were significantly lower in each stage than in the corresponding stage of induced labor. These results suggest that endogenous prostaglandin F2 alpha (PGF2 alpha) production decreases 2-3 weeks prior to the spontaneous onset of labor and is increased again as labor progresses, that the patterns of PGF2 alpha production are similar to each other during spontaneous labor and labor induced by various methods. Therefore, it is felt that endogenous PGF2 alpha may participate in the progress of all kinds of labor.     

Analysis of chromosome segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes

Individuals carrying translocations suffer from reduced fertility or spontaneous abortions and seek help in form of assisted reproductive technology (ART) and preimplantation genetic diagnosis (PGD). While most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are thus unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Thus several methods have been devised to detect translocation imbalance through FISH in single cells for purpose of PGD, among them polar body chromosome painting, interphase FISH with combination of subtelomeric and centromeric probes, breakpoint spanning probes, and cell conversion. Results with PGD indicate a significant decrease in spontaneous abortions, from 81% before PGD to 13% after PGD. They also indicate very high rates of chromosome abnormalities in embryos from translocation carriers, 72% for Robertsonian translocations and 82% for reciprocal translocations. Sperm analysis was found to be a good predictor of IVF and PGD outcome, with samples with more than 60% abnormal forms indicating poor prognosis. Similarly, the predictability from first PGD cycle results for future cycles was 90%. In summary, PGD can help translocation carriers to achieve viable pregnancies, but the success of the process is conversely related to the baseline of unbalanced gametes.     

Regulation of endothelial nitric oxide synthase by PGD(2) in the developing choroid

We investigated if prostaglandins might regulate the increased choroidal endothelial (e) nitric oxide synthase (NOS) expression in the perinate. Prostaglandins, eNOS mRNA, immunoreactive protein and activity, and nitrite [stable metabolite of nitric oxide (NO)] production were markedly higher in newborn (1 day old) than juvenile (6-8 wk old) pig choroid. Treatment of isolated newborn choroids with the prostaglandin synthase inhibitor ibuprofen for 24 h reduced eNOS mRNA and nitrite production to values in juveniles. This effect was equally observed with the PGD(2) receptor (DP) blocker BW A868C and was prevented by cotreatment with PGD(2) but not other prostaglandins; similar observations were made on NOS activity in vivo. PGD(2) also increased eNOS expression on choroids of juveniles, and this effect was blocked by BW A868C. The manifestation of this upregulation of eNOS by PGD(2) on the control of choroidal vasomotor response was tested by using NO-dependent vasorelaxants, ACh, bradykinin (Bk), and substance P (SP). ACh-, Bk-, and SP-elicited choroidal vasorelaxation was greater in saline-treated newborn than juvenile pigs. Ibuprofen (24 h) decreased ACh-, Bk-, and SP-evoked vasorelaxation in newborns, whereas PGD(2) increased that in juveniles and prevented the ibuprofen-induced attenuated relaxation in newborns; infusion of N(omega)-monomethyl-L-arginine in choroids of those animals treated with PGD(2) reversed the augmented vasorelaxation to ACh, Bk, and SP. Finally, PGD(2)-induced upregulation of NOS in the perinate was also reflected by curtailed choroidal blood flow autoregulatory response to increased perfusion pressure. In conclusion, PGD(2) exhibits a major role in upregulating eNOS expression and activity in the choroid, which in turn results in greater NO-mediated vasorelaxation; a new mechanism for eNOS regulation via DP is hereby disclosed. The relationship between PGD(2) and eNOS in the developing subject provides an explanation for the interactive role of these two factors in the absent choroidal blood flow autoregulation in the perinate.     

Cholinergic neuromodulation by prostaglandin D2 in canine airway smooth muscle

To determine whether prostaglandin D2 (PGD2) modulates cholinergic neurotransmission in airway smooth muscle and, if so, what the mechanism of action is, we studied bronchial segments from dogs under isometric conditions in vitro. PGD2 (10(-8)-10(-5) M) elicited dose-dependent muscle contraction, which was reduced after blockade of muscarinic receptors, so that 50% effective dose (ED50) increased from 1.3 +/- 0.3 X 10(-6) to 3.9 +/- 1.0 X 10(-6) M by atropine (10(-6) M) (mean +/- SE, P less than 0.05). Physostigmine, at a concentration insufficient to alter base-line tension (10(-8) M), enhanced the PGD2-induced contraction and decreased ED50 to 6.4 +/- 0.5 X 10(-7) M (P less than 0.05). When added at the highest doses that did not cause spontaneous contraction (1.9 +/- 0.5 X 10(-7) M), PGD2 increased the contractile response to electrical field stimulation (1-50 Hz) by 21.9 +/- 6.6% (P less than 0.001). In contrast to this effect, the response to administered acetylcholine was not affected by PGD2. On the other hand, PGD2-induced augmentation of the response to electrical field stimulation (5 Hz) was further increased from 23.6 +/- 3.0 to 70.4 +/- 8.8% in the presence of physostigmine (10(-8) M) and was abolished by atropine but not affected by the alpha-adrenergic antagonist phentolamine or the histamine H1-blocker pyrilamine. These results suggest that the contraction of airway smooth muscle induced by PGD2 is in in part mediated by a cholinergic action and that PGD2 prejunctionally augments the parasympathetic contractile response, likely involving the accelerated release of acetylcholine at the neuromuscular junction.     

Selective inhibition of arachidonic acid metabolite release from human lung tissue by antiinflammatory steroids

The effects of antiinflammatory steroids on arachidonic acid metabolite release from human lung fragments were analyzed. Incubation of lung fragments for 24 hr with 10(-6) M dexamethasone inhibited the net release of the prostacyclin metabolite 6-keto-PGF1 alpha, PGE2, and PGF2 alpha from lung fragments stimulated with anti-IgE but failed to inhibit the anti-IgE-induced release of PGD2, TXB2, and iLTC4. The IC50 of dexamethasone for inhibition of both spontaneous and anti-IgE-induced 6-keto-PGF1 alpha release was approximately 2 X 10(-8) M, and a 6-hr preincubation with the drug was required for 50% inhibition of prostaglandin release. Other agents were tested for activity in stimulating arachidonic acid metabolite release from human lung fragments. FMLP (fmet-leu-phe) stimulated the release of all metabolites tested (6-keto-PGF1 alpha, PGD2, PGE2, PGF2 alpha, TXB2, iLTC4); platelet-activating factor (PAF), but not lysoPAF, stimulated the release of PGD2, TXB2, and iLTC4. In contrast to the case with anti-IgE, where dexamethasone failed to inhibit net PGD2 and TXB2 release, the steroid inhibited the release of these metabolites stimulated by both FMLP and PAF. The steroid inhibited iLTC4 release induced by the highest concentration of PAF (10(-6)M) but did not inhibit iLTC4 release stimulated by either 10(-7) M PAF, FMLP, or anti-IgE. Because neither FMLP nor PAF caused the release of PGD2 or TXB2 from purified human lung mast cells, and because they also failed to induce histamine release from lung fragments, it is suggested that these stimuli produce PGD2 and TXB2 release in lung fragments through an action on a cell distinct from the mast cell. This suggestion is supported by the selective inhibition of the release of these arachidonic acid metabolites by dexamethasone. We suggest that the inhibitory action of steroids on arachidonic acid metabolite in human lung fragments contributes to their therapeutic efficacy in pulmonary diseases.     

Interleukin-1beta induces calcium transients and enhances basal and store operated calcium entry in human myometrial smooth muscle

We have previously reported increased protein expression of sarcoplasmic reticulum calcium ATPase (SERCA) 2b in myometrium from women in labor at term, but the stimulus for this change is unknown. Proinflammatory cytokines have been implicated in the cascade of events leading to preterm and term labor, and we hypothesize that interleukin (IL)-1beta may induce changes in key calcium homeostatic mechanisms and, in turn, augment myometrial contractility before labor. The aim of the present study was to investigate the long-term effects of IL-1beta on SERCA 2b protein expression, calcium mobilization from intracellular stores, and store-operated calcium entry. Myometrial biopsies were obtained, with consent, from women undergoing elective cesarean section at term. Primary-cultured human myometrial smooth muscle (HMSM) cells were exposed to IL-1beta (10 ng/ml) for 24 h or to culture medium alone (control). Cells were subsequently used in Western blot studies or loaded with fura-2 to assess calcium dynamics using fluorescent digital imaging. The present study clearly demonstrated that IL-1beta significantly increased SERCA 2b protein expression in HMSM cells. Cyclopiazonic acid-induced calcium transients were also augmented, predominantly by activation of lanthanum-sensitive, store-operated calcium entry. HMSM cell excitability was enhanced, as evidenced by increased basal calcium entry and the initiation of spontaneous calcium transients in 37% of IL-1beta-treated cells. IL-1beta modulation of calcium mobilization may be an important mechanism in the cascade of events preparing the pregnant uterus for labor.     

Cellular communication inside the liver. Binding, conversion and metabolic effect of prostaglandin D2 on parenchymal liver cells.

The major eicosanoid produced within the rat liver, prostaglandin (PG) D2, wa studied for its ability to interact with the various liver cell types. It appeared that PGD2 bound specifically to parenchymal liver cells, whereas the binding of PGD2 to Kupffer and endothelial liver cells was quantitatively unimportant. Maximally 700 pg of PGD2/mg of parenchymal-cell protein could be bound by a high-affinity site (1 x 10(6) PGD2-binding sites/cell). The recognition site for PGD2 is probably a protein because trypsin treatment of the cells virtually abolished the high-affinity binding. High-affinity binding of PGD2 was a prerequisite for the induction of a metabolic effect in isolated parenchymal liver cells, i.e. the induction of glycogenolysis. High-affinity binding of PGD2 by parenchymal cells was coupled to the conversion of PGD2 into three metabolites, whereas no conversion of PGD2 by Kupffer and endothelial liver cells was noticed. The temperature-sensitivity of the conversion of PGD2 was consistent with a conversion of PGD2 on or in the vicinity of the cell membrane. One of the PGD2 metabolites could be identified as 9 alpha, 11 beta-PGF2. It can be calculated that the conversion rate of PGD2 by parenchymal liver cells exceeds the production rate of PGD2 by Kupffer plus endothelial liver cells, indicating that PGD2 is meant to exert its activity within the liver. The present finding that PGD2 formed by the non-parenchymal liver cells is recognized by a specific receptor on parenchymal liver cells and that binding, conversion and metabolic effect of PGD2 are interlinked by this receptor provides further support for the specific role of PGD2 in the intercellular communication in the liver.     

Lipocalin-type prostaglandin D2 synthase protein regulates glial cell migration and morphology through myristoylated alanine-rich C-kinase substrate: prostaglandin D2-independent effects

Prostaglandin D synthase (PGDS) is responsible for the conversion of PGH(2) to PGD(2). Two distinct types of PGDS have been identified: hematopoietic-type PGDS (H-PGDS) and lipocalin-type PGDS (L-PGDS). L-PGDS acts as both a PGD(2)-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. Although L-PGDS is one of the most abundant proteins in the cerebrospinal fluid, little is known about the function of L-PGDS in the central nervous system (CNS). To better understand the role of L-PGDS in the CNS, effects of L-PGDS on the migration and morphology of glial cells were investigated. The L-PGDS protein accelerated the migration of cultured glial cells. Expression of the L-pgds gene was detected in glial cells and neurons. L-PGDS protein also induced morphological changes in glia similar to the characteristic phenotypic changes in reactive gliosis. L-PGDS-induced cell migration was associated with augmented formation of actin filaments and focal adhesion, which was accompanied by activation of AKT, RhoA, and JNK pathways. L-PGDS protein injected into the mouse brain promoted migration and accumulation of astrocytes in vivo. Furthermore, the cell migration-promoting effect of L-PGDS on glial cells was independent of the PGD(2) products. The L-PGDS protein interacted with myristoylated alanine-rich protein kinase C substrate (MARCKS) to promote cell migration. These results demonstrate the critical role of L-PGDS as a secreted lipocalin in the regulation of glial cell migration and morphology. The results also indicate that L-PGDS may participate in reactive gliosis in an autocrine or paracrine manner, and may have pathological implications in neuroinflammatory diseases.     

Proteomic analysis of human cervico-vaginal fluid displays differential protein expression in association with labor onset at term

Human labor is characterized by dramatic physiological and structural alterations of the cervix and overlying fetal membranes, leading to myometrial activation and delivery. To investigate the potential mechanism of these changes, we performed 2D PAGE proteomic analysis on serial cervico-vaginal fluid samples obtained from women during late pregnancy and spontaneous labor. We identified 9 protein spots that were significantly altered ( p < 0.05) in association with spontaneous term labor. Eight protein spots were definitively characterized by electrospray ion-trap mass spectrometry yielding 7 different proteins: cystatin-A, interleukin-1 receptor antagonist, glutathione S-transferase P, peroxiredoxin-2, thioredoxin, copper-zinc superoxide dismutase, and epidermal fatty-acid binding protein. These proteins are involved in protease inhibition, anti-inflammatory cytokine activity, and oxidative stress defense. These findings may provide an insight into the biochemical processes and timing associated with extracellular matrix remodelling of the cervix, supracervical fetal membranes, and myometrial activation in association with spontaneous term labor. Application of these findings may lead to development of predictive biomarkers of labor onset.     

Cutting edge: prostaglandin D2 enhances leukotriene C4 synthesis by eosinophils during allergic inflammation: synergistic in vivo role of endogenous eotaxin

In addition to the well-recognized ability of prostaglandin D2 (PGD2) to regulate eosinophil trafficking, we asked whether PGD2 was also able to activate eosinophils and control their leukotriene C4 (LTC4)-synthesizing machinery. PGD2 administration to presensitized mice enhanced in vivo LTC4 production and formation of eosinophil lipid bodies-potential LTC4-synthesizing organelles. Immunolocalization of newly formed LTC4 demonstrated that eosinophil lipid bodies were the sites of LTC4 synthesis during PGD2-induced eosinophilic inflammation. Pretreatment with HQL-79, an inhibitor of PGD synthase, abolished LTC4 synthesis and eosinophil lipid body formation triggered by allergic challenge. Although PGD2 was able to directly activate eosinophils in vitro, in vivo PGD2-induced lipid body-driven LTC4 synthesis within eosinophils was dependent on the synergistic activity of endogenous eotaxin acting via CCR3. Our findings, that PGD2 activated eosinophils and enhanced LTC4 synthesis in vivo in addition to the established PGD2 roles in eosinophil recruitment, heighten the interest in PGD2 as a target for antiallergic therapies.     

Development of enzyme-linked immunosorbent assay for prostaglandin D2 using the stable isosteric analogue as a hapten mimic and its application

For the determination of prostaglandin (PG) D(2) produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD(2) is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD(2). In an attempt to get a specific antibody for PGD(2), we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD(2), a novel, chemically stable, isosteric analogue of PGD(2). We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD(2). To develop the enzyme-linked immunosorbent assay (ELISA) for PGD(2), the immobilized antigen using the stable PGD(2) derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD(2). The optimization of the assay provided a sensitive calibration curve for PGD(2) from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD(2) was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD(2) in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD(2) in the maturation medium of adipocytes at 37 degrees C caused the chemical conversion into PGJ(2) derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD(2) before the conversion starts to occur.     

Prostaglandin D2 stimulates vasoactive intestinal polypeptide release into rat hypophysial portal blood

The effect of prostaglandin D2 (PGD2) on vasoactive intestinal polypeptide (VIP) release from the hypothalamus was examined by determining plasma VIP levels in rat hypophysial portal blood. Intraventricular injection of PGD2 (5 micrograms/rat) caused a 3-fold increase in the concentration of plasma VIP in hypophysial portal blood in anesthetized rats. A PGD2 metabolite, 13,14-dihydro-15-keto PGD2, did not affect VIP levels in portal blood. The flow rate of hypophysial portal blood was not changed after the injection of PGD2. The intraventricular injection of PGD2, but not PGD2 metabolite, resulted in an increase in peripheral plasma prolactin (PRL) levels in the rat. These findings suggest that PGD2 plays a stimulatory role in regulating VIP release from the hypothalamus into hypophysial portal blood and causes PRL secretion from the pituitary in rats.     

Induction of apoptosis in non-small cell lung carcinoma A549 cells by PGD₂ metabolite, 15d-PGJ₂

PGD2 (prostaglandin D2) is a mediator in various pathophysiological processes, including inflammation and tumorigenesis. PGD2 can be converted into active metabolites and is known to activate two distinct receptors, DP (PGD2 receptor) and CRTH2/DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). In the past, PGD2 was thought to be involved principally in the process of inflammation. However, in recent years, several studies have shown that PGD2 has anti-proliferative ability against tumorigenesis and can induce cellular apoptosis via activation of the caspase-dependent pathway in human colorectal cancer cells, leukaemia cells and eosinophils. In the lung, where PGD2 is highly released when sensitized mast cells are challenged with allergen, the mechanism of PGD2-induced apoptosis is unclear. In the present study, A549 cells, a type of NSCLC (non-small cell lung carcinoma), were treated with PGD2 under various conditions, including while blocking DP and CRTH2/DP2 with the selective antagonists BWA868C and ramatroban respectively. We report here that PGD2 induces A549 cell death through the intrinsic apoptotic pathway, although the process does not appear to involve either DP or CRTH2/DP2. Similar results were also found with H2199 cells, another type of NSCLC. We found that PGD2 metabolites induce apoptosis effectively and that 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2) is a likely candidate for the principal apoptotic inducer in PGD2-induced apoptosis in NSCLC A549 cells.     

Transformation of prostaglandin D2 to isomeric prostaglandin F2 compounds by human eosinophils. A potential mast cell-eosinophil interaction

PGD2 undergoes extensive isomerization in vivo followed by metabolism by 11-ketoreductase to yield a family of biologically active isomeric PGF2 compounds, including 9, alpha 11 beta-PGF2. Because immunologically activated human mast cells produce substantial quantities of PGD2 and eosinophils accumulate around mast cells at sites of immediate hypersensitivity reactions, the ability of eosinophils to metabolize PGD2 was investigated. Purified human circulating eosinophils from four different donors transformed PGD2 to 9, alpha 11 beta-PGF2 and 12-epi-9 alpha, 11 beta-PGF2 in a time- and concentration-dependent manner. The formation of these compounds increased rapidly during the first 30 min of incubation of eosinophils with PGD2 and tended to plateau at approximately 2 h. Detection and quantification of the formation of 9 beta,11 beta-PGF2 and its 12-epi isomer was accomplished by a negative ion chemical ionization gas chromatography/mass spectrometry assay. On one occasion, eosinophils from one donor also transformed PGD2 to two additional isomeric PGF2 compounds, the stereochemical structures of which were not identified. The ability of eosinophils to produce PGD2 was then investigated. After stimulation with 2 microM A23187, the major cyclooxygenase product formed was thromboxane B2 (2247 pg/10(6) eosinophils) whereas only small quantities of PGD2 were produced (50 pg/10(6) eosinophils). Inasmuch as PGF2 compounds can exert biologic actions that differ from those of PGD2, this ability of eosinophils to transform PGD2 to PGF2 compounds could alter the local biologic effects of PGD2 released from adjacent mast cells and thus may represent a physiologically relevant mast cell-eosinophil interaction.     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

Mechanism of prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts

We previously showed that prostaglandin D(2) (PGD(2)) stimulates activation of protein kinase C (PKC). We investigated whether PGD(2) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD(2) increased the levels of HSP27 while having little effect on HSP70 levels. PGD(2) stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 microM. PGD(2) induced an increase in the levels of mRNA for HSP27. The PGD(2)-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD(2) induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD(2) was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD(2)-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD(2)-increased levels of mRNA for HSP27. These results strongly suggest that PGD(2) stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.     

Eosinophils as a novel cell source of prostaglandin D2: autocrine role in allergic inflammation

PGD(2) is a key mediator of allergic inflammatory diseases that is mainly synthesized by mast cells, which constitutively express high levels of the terminal enzyme involved in PGD(2) synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD(2). PGD(2) synthesis was evaluated within human blood eosinophils, in vitro differentiated mouse eosinophils, and eosinophils infiltrating inflammatory site of mouse allergic reaction. Biological function of eosinophil-derived PGD(2) was studied by employing inhibitors of synthesis and activity. Constitutive expression of H-PGDS was found within nonstimulated human circulating eosinophils. Acute stimulation of human eosinophils with A23187 (0.1-5 μM) evoked PGD(2) synthesis, which was located at the nuclear envelope and was inhibited by pretreatment with HQL-79 (10 μM), a specific H-PGDS inhibitor. Prestimulation of human eosinophils with arachidonic acid (10 μM) or human eotaxin (6 nM) also enhanced HQL-79-sensitive PGD(2) synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C(4) synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD(2) in response to arachidonic acid stimulation. In vivo, at late time point of the allergic reaction, infiltrating eosinophils found at the inflammatory site appeared as an auxiliary PGD(2)-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD(2), hence representing during allergic inflammation an extra cell source of PGD(2), which functions as an autocrine signal for eosinophil activation.