Eosinophils Are Potent Activators of Human 2J And2 -Prostaglandins D

15-Deoxy-∆ 12,1412,14 -prostaglandins D2 and J 2 Are Potent Activators of Human Eosinophils

This information is current as Guillaume Monneret, Hongping Li, Julian Vasilescu, Joshua of October 1, 2021. Rokach and William S. Powell J Immunol 2002; 168:3563-3569; ; doi: 10.4049/jimmunol.168.7.3563 http://www.jimmunol.org/content/168/7/3563 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ⌬12,14 15-Deoxy- -prostaglandins D2 and J2 Are Potent Activators of Human Eosinophils1

Guillaume Monneret,2* Hongping Li,* Julian Vasilescu,* Joshua Rokach,† and William S. Powell3*

⌬12,14 15-Deoxy- -PDJ2 (15d-PGJ2) is a degradation product of PGD2 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-␥. 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 ϳ on neutrophils or monocytes. 15d-PGJ2 has an EC50 of 10 nM, similar to that of its precursor, PGD2. The concentrations of

15d-PGJ2 required to activate eosinophils are thus much lower than those required for its anti-inﬂammatory effects (usually

⌬12,14 Downloaded from micromolar). 15-Deoxy- -prostaglandin D2 (15d-PGD2) is also a potent activator of eosinophils, with an EC50 about the same ⌬12 as that of PGD2, whereas -PGJ2 is slightly less potent. Eosinophils pretreated with PGD2 no longer respond to 15d-PGJ2, 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-PGJ2 are mediated by the DP2/chemoattractant receptor-homologous molecule expressed on Th2 cells that has recently been identified in eosinophils. 15d-PGJ2 is selective for the DP2 receptor, in that it has no effect on DP1 receptor-mediated adenylyl cyclase activity in platelets. We conclude that 15d-PGJ2 and 15d-PGD2 are selective http://www.jimmunol.org/ DP2 receptor agonists that activate human eosinophils with potencies at least 100 times greater than those for the proposed anti-inflammatory effects of 15d-PGJ2 on other cells. The Journal of Immunology, 2002, 168: 3563–3569. ␥ rostaglandin D2 undergoes degradation to 15-deoxy- inflammatory effects due to stimulation of PPAR may be due to ⌬12,14 4 ␬ -PGJ2 (15d-PGJ2), which has been proposed as an antagonism of the transcription factors NF- B, AP-1, and STAT-1 anti-inflammatory compound because of its various inhib- (6). 15d-PGJ also has anti-inflammatory effects that are indepen- P 2 itory effects, some of which are mediated by peroxisome prolif- dent of PPAR␥. It inhibits adhesion and the oxidative burst in erator-activated receptor-␥ (PPAR␥). Most notably, it strongly ac- neutrophils by a mechanism that appears not to be mediated by any tivates PPAR␥ (1, 2), a transcription factor that is preferentially known receptors (9). At least some non-PPAR␥-mediated anti- by guest on October 1, 2021 expressed in adipose tissue, vascular smooth muscle cells, and inflammatory effects of 15d-PGJ2 are due to the chemical reactiv- macrophages (3). PPAR␥ plays a central role in adipogenesis, en- ity of its dienone system, resulting in addition to protein thiol hances sensitivity to insulin, and inhibits the inflammatory re- groups, as has been shown for NF-␬B (10Ð12). The anti- ␥ sponse (3). Stimulation of PPAR by relatively high (low micro- inflammatory effects of 15d-PGJ2 described above are not shared ␥ molar) concentrations of 15d-PGJ2, as well as by other PPAR by its precursor PGD2. agonists results in suppression of the expression of cytokines (4, Little information is available about the ability of 15d-PGJ2 to stim-

5), matrix metalloproteinase-9, and inducible nitric oxide synthase ulate PGD2 receptors, and the effects of this substance on eosinophils ␥ (6). In addition, activation of PPAR induces apoptosis in a variety have not previously been explored. 15d-PGJ2 has been reported to of cells including macrophages (7, 8). Many of the anti- compete only very weakly for binding to the DP1 receptor that is coupled positively to adenylyl cyclase (13). We have recently identified a second receptor for PGD (DP receptor) on eosinophils, ac- *Meakins-Christie Laboratories, Department of Medicine, McGill University, Mon- 2 2 treal, Quebec, Canada; and †Claude Pepper Institute and Department of Chemistry, tivation of which leads to cell migration, increased expression of Florida Institute of Technology, Melbourne, FL 32901 CD11b, shedding of L-selectin, and actin polymerization (14). This Received for publication November 20, 2001. Accepted for publication February receptor appears to be identical with chemoattractant receptor-homol- 1, 2002. ogous molecule expressed on Th2 cells (CRTH2), an orphan receptor The costs of publication of this article were defrayed in part by the payment of page for which PGD was recently identified as a ligand (15). We previ- charges. This article must therefore be hereby marked advertisement in accordance 2 with 18 U.S.C. Section 1734 solely to indicate this fact. ously found that PGJ2 stimulates both DP1 and DP2 receptors on 1 This work was supported by the Canadian Institutes for Health Research Grant eosinophils, indicating that the 9-hydroxyl group is not required for MT-6254 (to W.S.P.), the J. T. Costello Memorial Research Fund, National Institutes biological activity (14). Furthermore, 13,14-dihydro-15-oxo-PGD2 is of Health Grant DK44730 (to J.R.), and National Science Foundation Grant CHE- a good agonist at the DP but not the DP receptor, indicating that 90-13145 for an AMX-360 NMR instrument (to J.R.). 2 1 although the 15-hydroxyl group of PGD is required for activation of 2 Current address: Flow Cytometry Unit, Immunology Laboratory, Lyon-Sud Uni- 2 versity Hospital, 69495 Perre-Be«nite, France. the DP1 receptor, it is not essential for interaction with the DP2 re- 3 Address correspondence and reprint requests to Dr. William S. Powell, Meakins- ceptor (14, 15). This raised the possibility that 15d-PGJ2, which lacks Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain this hydroxyl group, could have proinflammatory effects by selec- Street, Montreal, Quebec, Canada H2X 2P2. E-mail address: William. [email protected] tively stimulating the DP2 receptor in inflammatory cells. The present 4 ⌬12,14 Abbreviations used in this paper: 15d-PGJ2, 15-deoxy- -PGJ2; 15d-PGD2, 15- study was designed to examine the effects of 15d-PGJ on eosinophils ⌬12,14 ␥ ␥ 2 deoxy- -PGD2; PPAR , peroxisome proliferator-activated receptor- ; 5-oxo- ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; CRTH2, chemoattractant receptor-ho- and to determine whether it could stimulate these cells by activating mologous molecule expressed on Th2 cells. the DP2 receptor.

Materials and Methods (2 ml/106 cells) was then added, and the mixture was centrifuged at 200 ϫ g for 10 min. The pellet was resuspended in ice cold PBS containing Ca2ϩ Materials ϩ (1.8 mM) and Mg2 (1 mM) to give a concentration of 5 ϫ 106 cells/ml. All prostaglandins were purchased from Cayman Chemical (Ann Arbor, Aliquots of the leukocyte suspension (90 ␮l for the concentration-response Ն ␮ MI). 15d-PGJ2 was the purified 14-cis isomer ( 97% pure; catalog number experiment; 800 l for the time course experiment) were preincubated for 18570). 15-Deoxy-⌬12,14-prostaglandin D (15d-PGD ) is the 14-trans iso- 5 min at 37¡C before the addition of agonist or vehicle (10 ␮l PBS con- 2 2 ϩ ϩ mer. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) was synthesized taining Ca2 and Mg2 and 0.1% BSA). The incubations were terminated chemically as described previously (16). at various times (20 s for the concentration-response experiment) by addition of formaldehyde (37%) to give a final concentration of 8.5%. After Preparation of leukocytes the samples were kept on ice for 30 min, a mixture of lysophosphatidyl- choline (30 ␮g in 23.8 ␮l PBS) and N-(7-nitrobenz-2-oxa-1,3-diazol-4- All studies with eosinophils were conducted using unfractionated leuko- yl)phallacidin (Molecular Probes; 49 pmol in 6.2 ␮l methanol; final con- cytes from healthy volunteers. Whole blood was treated with Dextran centration, 0.3 ␮M) was added to each sample (18), followed by incubation T-500 (Amersham-Pharmacia Biotech, Piscataway, NJ) for 45 min at 4¡C overnight in the dark at 4¡C. The cells were then washed by addition of ϫ to remove RBC (17). The supernatant was centrifuged at 200 g for 10 PBS (1 ml), followed by centrifugation at 200 ϫ g for 10 min and resus- min, and the pellet was suspended in water (10 ml). After 30 s, the suspension of the pellet in 300 ␮l PBS containing 1% formaldehyde. F-actin ϫ pension was diluted with 2 concentrated PBS to give final concentrations levels were measured by flow cytometry in eosinophils (high side scatter; of 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4 low CD16) and neutrophils (high side scatter; high CD16). at pH 7.4. For experiments designed to measure CD11b expression, the cells were resuspended directly in PBS without prior lysis of RBC (this was Measurement of CD11b expression accomplished later using OptiLyse (Beckman Coulter, Fullerton, CA)). Leukocytes (0.5 ml; 106/ml) in PBS containing Ca2ϩ and Mg2ϩ were in- Preparation of platelets cubated with agonists for 10 min. The incubations were terminated by the

addition of ice cold FACSFlow (BD Biosciences) and centrifugation. The Downloaded from Whole blood (20 ml) was collected in medium (2.8 ml) containing citric cells were then incubated for 30 min at 4¡C with a mixture of PE-labeled acid (15.5 mM), sodium citrate (90 mM), NaH2PO4 (16 mM), dextrose mouse anti-human VLA-4 (5 ␮l; BD Biosciences) and FITC-labeled (161 mM), and adenine (2 mM). After centrifugation at 200 ϫ g for 15 mouse anti-human CD11b (10 ␮l Bear1; Beckman Coulter). After incu- min, the supernatant was diluted with an equal volume of medium con- bation with OptiLyse C (0.25 ml; Beckman Coulter) for 15 min, the cells taining 94 mM citrate and 140 mM dextrose, pH 6.5. The mixture was were centrifuged and fixed in PBS (0.4 ml) containing 1% formaldehyde. centrifuged at 1000 ϫ g for 10 min, and the pellet was suspended in PBS The distribution of fluorescence intensities among 60,000 cells was mea- ϩ ϩ containing Ca2 (1.8 mM) and Mg2 (1 mM) to give a platelet concen- sured by flow cytometry. CD11b was measured in both eosinophils (high tration of 3 ϫ 108 cells/ml. side scatter; high VLA-4) and neutrophils (high side scatter; low VLA-4) http://www.jimmunol.org/ (19). The cells gated as eosinophils contained Ͻ2% basophils, based on Analysis of intracellular calcium levels by flow cytometry expression of high IgE. Leukocytes (107 cells/ml) were treated with the acetoxymethyl ester of Determination of cAMP levels in platelets fluo-3 (2 ␮M; Molecular Probes, Eugene, OR) in the presence of Pluronic F-127 (0.02%; Molecular Probes) for 60 min at 23¡C. The mixture was Platelets (3 ϫ 107 cells in 100 ␮l) were incubated with prostanoids for 2 then centrifuged at 200 ϫ g for 10 min, and the pellet was resuspended in min at 37¡C. The incubations were terminated by addition of ice cold PBS to give a concentration of 50 ϫ 106 cells/ml. The leukocytes were then ethanol (300 ␮l), and the precipitated proteins were removed by centrifu- treated with PC5-labeled mouse anti-human CD16 (3.3 ␮l/106 cells; Beck- gation (600 ϫ g for 15 min). cAMP in the supernatants was measured using man Coulter) for 30 min at 0¡C. PBS (25 ml) was then added, the mixture a competitive protein-binding radiometric assay (Diagnostic Products, Los

centrifuged as described above, and the pellet was resuspended in PBS to Angeles, CA) according to the manufacturer’s instructions. by guest on October 1, 2021 give a concentration of 3 ϫ 106 leukocytes/ml. After incubation at 23¡C for 30 min, an aliquot (0.95 ml) of the leukocyte suspension was removed and Results ␮ 2ϩ 2ϩ treated with PBS (50 l) containing Ca (36 mM) and Mg (20 mM). 15d-PGJ2 is a potent and selective stimulator of calcium After 5 min, the cells were analyzed by flow cytometry using a FACS- mobilization in eosinophils Calibur instrument (BD Biosciences, San Jose, CA). A total of ϳ106 cells was counted during a period of 3Ð4 min for each sample. Fluo-3 fluores- Intracellular calcium levels were measured by flow cytometry in cence was measured in eosinophils, neutrophils, and monocytes, which mixed leukocytes loaded with fluo-3 and treated with PC5-tagged were gated out on the basis of CD16 staining and side scatter (Fig. 1A). anti-CD16, which labeled neutrophils but not eosinophils. This Maximal calcium responses were determined by addition of A23187 (10 ␮M) at the end of each run. averted the potential for eosinophil activation that could occur if an Ab to an eosinophil surface marker were used. Eosinophils (very Measurement of actin polymerization low CD16; high side scatter) could easily be distinguished from Leukocytes (5 ϫ 107 cells/ml) were treated with PC5-labeled mouse anti- neutrophils (high CD16; high side scatter) and monocytes (low ␮ 6 human CD16 (10 l/10 cells; Beckman Coulter) for 30 min on ice. PBS CD16; low side scatter) (Fig. 1A). The effects of 15d-PGJ2 on

FIGURE 1. Effects of 15d-PGJ2 on intracellular calcium levels in eosinophils, neutrophils, and monocytes. Leukocytes were loaded with fluo-3, stained with PC5-labeled anti-CD16, and analyzed by flow cytometry. A, Dot plot showing the separation of eosinophils (Eos), neutrophils (Neutr), and monocytes (Mono) by flow cytometry on the basis of side scatter and CD16 staining. B, Changes in fluo-3 fluorescence induced in eosinophils (bottom), neutrophils ␮ (middle), and monocytes (top) by addition of 5-oxo-ETE (5o; left), 15d-PGJ2 (15dJ2; center), and PGD2 (D2; right), all at final concentrations of 1 M. E F C, Concentration-response curves for the effects of PGD2 ( ) and 15d-PGJ2 ( ) on calcium mobilization in eosinophils. The values are expressed as Ϯ percentages of the maximal responses to PGD2 (649 73 nM above baseline) and are means of experiments on leukocytes from four different donors. The Journal of Immunology 3565

intracellular calcium levels in the above three cell types were com-

pared with those of PGD2 and 5-oxo-ETE (Fig. 1B). PGD2 stimulates calcium mobilization (20) and a variety of other responses (14) in eosinophils but not neutrophils, whereas 5-oxo-ETE acti- vates both eosinophils and neutrophils (21Ð23). This is conﬁrmed

in the present study. We also found that neither PGD2 nor 5-oxo- ETE affects calcium levels in monocytes (Fig. 1B, top tracings). ␮ As with PGD2, 15d-PGJ2 (1 M) induced calcium mobilization selectively in eosinophils, but not in either neutrophils or monocytes. The concentration-response curves for calcium mobilization Ϯ Ϯ induced by 15d-PGJ2 (EC50 29 15 nM) and PGD2 EC50 60 47 nM) were similar to one another, although there was consider- able variability in the magnitude of the individual responses using this method (Fig. 1C).

15d-PGJ2 selectively stimulates actin polymerization in eosinophils

The effects of 15d-PGJ2 on actin polymerization in eosinophils

were compared with those of PGD2 as well as its more immediate Downloaded from ⌬12 precursors, PGJ2 and -PGJ2 (Fig. 2). Actin polymerization was measured in unfractionated leukocytes using anti-CD16-labeling to distinguish between eosinophils and neutrophils as shown in Ϯ Fig. 1A. 15d-PGJ2 (EC50 11 3 nM) was only slightly less potent FIGURE 3. Effects of prostaglandins on CD11b expression by eosino- Ϯ ⌬12 Ϯ than PGD2 (EC50 7 2 nM). -PGJ2 (EC50 23 2 nM) and phils. Human leukocytes were incubated with various prostaglandins for 10 Ϯ PGJ2 (EC50 30 2 nM) were somewhat less potent than 15d-PGJ2 min at 37¡C followed by ﬁxation with formaldehyde and staining with http://www.jimmunol.org/ ( p Ͻ 0.05). All four compounds induced similar maximal re- FITC-labeled anti-CD11b and PE-labeled anti-VLA-4. Eosinophils were sponses. In contrast to eosinophils, neither 15d-PGJ nor PGD gated out based on high side scatter and high VLA-4. The values are ex- 2 2 Ϯ induced actin polymerization in neutrophils (indicated by (N) in pressed as percentages of the maximal responses to PGD2 and are means Fig. 2). SE of determinations on leukocytes from the numbers of different donors indicated below. A, Effects of PGD2 dehydration products on CD11b ex- E ϭ F ϭ Œ 15d-PGJ and 15d-PGD are potent stimulators of CD11b pression: PGD2 ( ;D2; n 14), 15d-PGJ2 ( ;dJ2; n 13), 15d-PGD2 ( ; 2 2 ϭ ⌬12 f ⌬12 ϭ ‚ ϭ dD2; n 6), -PGJ2 ( ; ; n 9), and PGJ2 ( ; n 10). B, Effects expression by eosinophils E ϭ Œ ϭ ‚ ϭ of other prostaglandins: PGD2 ( ; n 6), PGD3 ( ; n 6), PGD1 ( ; n ␤ f ␤ ϭ ƒ ϭ CD11b expression was measured in mixed leukocytes by ﬂow cy- 6), 11 -PGF2␣ ( ;11 F2; n 3), and PGD1 alcohol ( ;hD1; n 3).

tometry. After incubation with agonists, the eosinophils were ﬁxed by guest on October 1, 2021 and stained with PE-labeled anti-VLA-4 to distinguish them from ⌬12 neutrophils. The effects of various dehydration products of PGD2 tencies. PGJ2 and -PGJ2 had similar effects but were slightly Ϯ ϳ on CD11b expression are shown in Fig. 3A. PGD2 (EC50, 9.4 less potent, with EC50 values of 30 nM. None of the prostanoids Ϯ 2.2 nM), 15d-PGJ2 (EC50, 11.7 2.7 nM), and 15d-PGD2 (EC50, shown in Fig. 3A stimulated CD11b levels in neutrophils (data not 8.0 Ϯ 2.5 nM) all stimulated CD11b expression with similar po- shown).

15d-PGJ2 does not act on DP1 receptors

To determine whether 15d-PGJ2 could activate DP1 receptors, we compared its effect to that of PGD2 on cAMP levels in human

platelets (Fig. 4). Consistent with published data (24), PGD2 strongly stimulated cAMP production in platelets. In contrast, 15d-

PGJ2 had no detectable effect on cAMP levels in these cells. This

FIGURE 2. Concentration-response curves for the effects of prostaglandins on actin polymerization in eosinophils. Leukocytes were stained with E ƒ ϭ PC5-labeled anti-CD16 and incubated with PGD2 ( , ; n 6), 15d-PGJ2 F ϭ ⌬12 f ⌬12 ϭ ⌬ ϭ ( , ; 15dJ2; n 6), -PGJ2 ( ; ; n 4), or PGJ2 ( ; n 4) for 20 s, followed by ﬁxation with formaldehyde and staining of F-actin with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin-labeled phallacidin. Eo- sinophils were gated out on the basis of high side scatter and minimal ƒ staining with anti-CD16. The effects of PGD2 ( ;D2 (N)) and 15d-PGJ2 FIGURE 4. Effects of 15d-PGJ2 and PGD2 on cAMP levels in human ( ; 15dJ2 (N)) on F-actin levels in neutrophils are shown for comparison. platelets. Platelets were incubated with various concentrations of 15d-PGJ2 F E The values are the percent increase in F-actin levels compared with vehi- ( ; 15dJ2)orPGD2 ( ) for 2 min at 37¡C and cAMP levels determined cle-treated controls and are means Ϯ SE of determinations on leukocytes using a competitive binding assay. The values are means Ϯ SE of deter- from the numbers of different donors indicated above. minations on platelets from three different individuals. 3566 15d-PGs ACTIVATE HUMAN EOSINOPHILS

ϳ is consistent with a previous study showing that 15d-PGJ2 is 300 times less potent than PGD2 in competing for binding to HEK 293 cells transfected with DP1 receptors (13).

15d-PGJ2 desensitizes eosinophils to PGD2 but not to 5-oxo-ETE

The similarity of the responses induced by PGD2 and 15d-PGJ2 in eosinophils and the lack of response of neutrophils to both agonists would be consistent with their interaction with the same receptor.

To further address this issue, we determined whether 15d-PGJ2 could desensitize eosinophils to the effects of PGD2. For this pur- pose, we examined two very rapid and transient responses, calcium mobilization and actin polymerization. Measurement of calcium mobilization has the advantage that it can be accomplished in real ␮ time. 15d-PGJ2 (1 M) induced a strong calcium response in eosinophils, as shown in Fig. 5A (tracings on left). However, eosin-

ophils treated with 15d-PGJ2 were completely unresponsive to ␮ PGD2 (1 M; Fig. 5A, upper left). Similarly, prior treatment of eosinophils with PGD desensitized them to 15d-PGJ (Fig. 5A,

2 2 Downloaded from upper middle). The selective DP1 receptor agonist BW245C, which on its own had no effect on calcium levels, did not alter the

response to 15d-PGJ2 (Fig. 5A, lower middle). In contrast to its inhibitory effect on PGD2-induced calcium mobilization, 15d- PGJ2 did not affect the response to 5-oxo-ETE. Neither was 5-oxo- ␮ ETE (1 M) able to prevent the response to either 15d-PGJ2 (Fig. http://www.jimmunol.org/ 5A, upper right)orPGD2 (Fig. 5A, lower right). We also determined whether 15d-PGJ2 could desensitize eosinophils to PGD2-induced actin polymerization. Leukocytes were ﬁrst treated with 15d-PGJ2 (100 nM), followed by addition of either PGD2 (100 nM) or 5-oxo-ETE (100 nM) 6 min later (Fig. 5B). F-actin levels were measured by ﬂow cytometry immediately be-

fore and at several times after addition of 15d-PGJ2. 15d-PGJ2 strongly stimulated actin polymerization in eosinophils, inducing a ϳ maximal response after 20 s. This declined by 50% by 6 min. by guest on October 1, 2021

Addition of PGD2 at that time had no effect on polymerized actin levels in eosinophils, whereas addition of 5-oxo-ETE resulted in a strong and rapid response, comparable with that induced by FIGURE 5. Cross-desensitization between 15d-PGJ2 and PGD2 but not 15d-PGJ2. 5-oxo-ETE. A, Fluo-3-loaded leukocytes were stained with PC5-labeled

anti-CD16 and analyzed by flow cytometry. Addition of 15d-PGJ2 (15d) at Effects of other structural modifications of PGD2 on eosinophil the time indicated by the arrow induced calcium mobilization and desen- activation sitized eosinophils to PGD2 (D2) but not to 5-oxo-ETE (5o) (top left and bottom left, respectively). PGD2 (top center), but not the specificDP1 re- We also examined the effects of other PGD2 analogs on CD11b expression in eosinophils (Fig. 3B). Introduction of a ⌬17 double ceptor agonist BW245C (BW; bottom center) desensitized eosinophils to 15d-PGJ . 5-Oxo-ETE did not prevent calcium mobilization in response to bond had relatively little effect on biological potency, given that 2 either 15d-PGJ (top right)orPGD (bottom right). Final concentrations of Ϯ ⌬5 2 2 PGD3 has an EC50 of 10 2 nM. Reduction of the double bond agonists were 1 ␮M in all cases. The results are representative of experi- Ϯ has a greater effect, increasing the EC50 to 92 40 nM (PGD1). ments with similar results using leukocytes from three different donors. B, However, this may be an underestimate, because the maximal re- Anti-CD16-PC5-labeled eosinophils were incubated with 15d-PGJ2 F sponse may not have been reached at the highest concentration (15dJ2; 100 nM), followed 6 min later by addition of either PGD2 ( )or used. The presence of the 11-oxo group is essential for activation 5-oxo-ETE (5oETE; E). Cells were fixed with formaldehyde either imme- ␤ diately before addition of 15d-PGJ or after 0.33, 3, 6 (immediately before of the DP2 receptor, because the reduction product 11 -PGF2␣ is 2 Ͼ100 times less potent than PGD . The carboxyl group also ap- addition of the second agonist), 6.33, and 10 min. The results are percent- 2 Ϯ pears to be required for activation of eosinophils via the DP re- age increases in F-actin over baseline and are means SE of values from 2 leukocytes from three different donors. ceptor, in that the 1-hydroxyl derivative of PGD1 (PGD1 alcohol; 1,9␣,15s-trihydroxyprost-13E-en-11-one) had no detectable effect on CD11b expression in these cells.

dienone system (10, 12, 28). The stimulatory effects of 15d-PGJ2 ϳ Discussion on eosinophils (EC50 10 nM) occur at substantially lower con- In this study, we have demonstrated that 15d-PGJ2 is a potent centrations than those required for its anti-inflammatory effects activator of human eosinophils, stimulating calcium mobilization, (generally low micromolar). Moreover, its effects on eosinophils actin polymerization, and CD11b expression. These proinflamma- are very rapid, requiring only a few seconds in the case of calcium tory effects are in contrast to a variety of anti-inflammatory effects mobilization and actin polymerization. In contrast, the anti-

(4, 25, 26) reported for this compound, which appear to be medi- inﬂammatory effects of 15d-PGJ2 are usually observed over a pe- ated by two major mechanisms: activation of PPAR␥ (27, 28); and riod of several hours. It would therefore seem unlikely that either

direct interaction with protein thiol groups as a result of its reactive of these mechanisms could explain the effects of 15d-PGJ2 on The Journal of Immunology 3567

eosinophils. Furthermore, the anti-inﬂammatory effects of 15d-

PGJ2 are not shared by its precursor PGD2, whereas this PG is also a potent activator of eosinophils. The selectivity of 15d-PGJ2 for eosinophils compared with neutrophils and monocytes also sug- gests a more speciﬁc mechanism of action than observed for its anti-inﬂammatory effects.

Although nearly all of the published studies on 15d-PGJ2 have focused on its anti-inflammatory properties, Zhang et al. (49) have recently shown that this compound has differential effects on chemokine production by monocytes, suppressing monocyte chemoattractant protein-1 release, but enhancing the production of IL-8. RANTES expression was unaffected. These effects were mediated ␥ by PPAR and required micromolar concentrations of 15d-PGJ2 and prolonged incubation times. In contrast to 15d-PGJ2, 15d- PGD2 had no effect on IL-8 secretion by monocytes. This study thus demonstrates that 15d-PGJ2 has the potential to induce both pro- and anti-inflammatory responses. However, the concentration dependence, structural requirements, and time dependence for these responses are quite different from the rapid and potent stim- FIGURE 6. Effects of structural modifications of PGD2 on biological Downloaded from ulatory effects of 15d-PGJ2 on eosinophils observed in the present activity. Filled arrows: pathways for the formations of dehydration/isomer- study. ization products; open arrows: other metabolic pathways and structural modifications. The parts of the structure that were altered are shaded. Val- The similarity between the effects of 15d-PGJ2 and PGD2 on eosinophils would suggest that the former compound might act by ues are the potency ratios for the stimulatory effects of PGD2 analogs on stimulating DP receptors on these cells. This issue was addressed CD11b expression compared with that of PGD2, for which the ratio would 2 be 1.0. They were calculated by dividing the EC for PGD by that of the by performing a series of cross-desensitization experiments. The 50 2

analog. Because there was some variability in the EC of PGD among http://www.jimmunol.org/ selective cross-desensitization between 15d-PGJ and PGD , but 50 2 2 2 experiments, the value for PGD was calculated separately for each analog, not with another eicosanoid proinﬂammatory mediator, 5-oxo- 2 so that only data for PGD2 obtained in the same experiments as those used ETE, provides compelling evidence that the actions of 15d-PGJ 2 to calculate the EC50 for the analog were used. For example, in the case of on eosinophils are indeed mediated by the DP2 receptor. The DP2- 15d-PGD2, the data for PGD2 were taken only from the six experiments used mediated proinﬂammatory effects of 15d-PGJ2 could therefore po- to calculate the EC50 for the former compound. The numbers of experiments tentially be observed only in cells possessing this receptor, which for each of the analogs can be found in the legend to Fig. 3. The results for include eosinophils (14, 15), basophils, and Th2 cells (15). 13,14-dihydro-15-oxo-PGD2 (dho-PGD2) were taken from Ref. 14.

Whether or not 15d-PGJ2 can also activate the latter two types of

cells remains to be determined. In contrast, the anti-inﬂammatory by guest on October 1, 2021 effects of this compound, although requiring substantially higher period of ϳ1 year, suggesting that storage is not a major problem. concentrations, can be observed in a wide variety of cells. Another possibility could be that the receptor responsible for the

The physiological signiﬁcance of 15d-PGJ2 is under debate (30, action of 15d-PGJ2 is not identical with the DP2/CRTH2 receptor, 31). It is a degradation product of PGD2 that can be formed spon- or that the DP2 receptor and CRTH2 are different from one an- taneously or in the presence of albumin, so that it could potentially other. This scenario would require the presence of two distinct

occur in vivo (32). The ﬁrst step in its formation is loss of the PGD2 receptors on eosinophils (i.e., a third DP receptor), both of

9-hydroxyl group of PGD2, which is followed by rearrangement of which mediate cell activation, and only one of which would be ⌬13 ⌬12 the double bond to give -PGJ2 and subsequent loss of the highly sensitive to 15d-PGJ2. Although we cannot completely ex- 15-hydroxyl group (Fig. 6). As yet, there is no evidence for its clude this possibility, it would seem rather unlikely in view of the

formation by an enzymatic process (33). However, substantial lev- fact that 15d-PGJ2 completely desensitizes eosinophils to PGD2,

els of 15d-PGJ2 have been detected by mass spectrometry in hu- suggesting that a single DP receptor on eosinophils is responsible man urine (34) and in pleural exudate ﬂuid after administration of for activation of these cells by both 15d-PGJ2 and PGD2. More-

carrageenin to rats (25). 15d-PGJ2 immunoreactivity has also recently over, other structural requirements for interaction with CRTH2 and been detected in foam cells in human atherosclerotic lesions as well as the DP2 receptor are quite similar to one another: 13,14-dihydro- in RAW 264.7 cells, a murine macrophage-like cell line (35). Even 15-oxo-PGD2 is a very good ligand for both receptors; the DP1

less is known about the physiological signiﬁcance of 15d-PGD2,al- receptor agonist BW245C is inactive at both receptors (14, 15), as ␤ though this compound is formed in much larger amounts than 15d- is the PGD2 metabolite 11 -PGF2␣, as shown both by the present

PGJ2 after incubation of PGD2 with albumin (32). study and by Hirai et al. (15). In addition, neither receptor is ac- ϳ In the present study, we found that 15d-PGJ2 (EC50 11 nM) tivated by any prostanoids unrelated to PGD2. Finally, it is possi- ϳ is nearly as potent as PGD2 (EC50 8 nM) in stimulating actin ble that the binding properties of DP2/CRTH2 ligands are different polymerization and CD11b expression in eosinophils, and at least in transfected K562 cells than in human eosinophils. This hypoth- as potent in inducing calcium mobilization. In contrast, Hirai et al. esis is difﬁcult to address directly in view of severe limitations in

(15) reported that 15d-PGJ2 has a Ki of 2300 nM compared with 61 the numbers of available eosinophils. However, it is conceivable 3 nM for PGD2 in displacing [ H]PGD2 from speciﬁc binding sites that K562 cells may lack some of the G proteins present in eosin- in K562 cells transfected with CRTH2. There are several possible ophils, which may result in differences in the conformation of the

explanations for this discrepancy. First, 15d-PGJ2 is a rather un- receptor that could affect the structural requirements for binding. ⌬12 stable molecule that exists in several isomeric forms, of which the We also observed a similar discrepancy for -PGJ2, which ␻ 14-cis form is the most abundant (33). Although it is possible that differs from PGJ2 only in the position of the double bond in the ⌬12 this instability could lead to altered biological responses, in our side chain of the molecule. -PGJ2 has cytotoxic effects on a ϳ ␮ hands responses to 15d-PGJ2 have been quite consistent over a variety of tumor cell lines (EC50 1 M) (36). In the present 3568 15d-PGs ACTIVATE HUMAN EOSINOPHILS

study, we found that this compound is approximately as potent as would suggest that activation of this receptor could be much more

PGJ2 in stimulating CD11b expression and actin polymerization prolonged than that of other prostanoid receptors, which would ϳ ⌬12 (EC50 25Ð30 nM). In contrast, -PGJ2 was reported to have a cease to be stimulated after the rapid biological inactivation of 3 Ki of 7100 nM in competing with [ H]PGD2 for binding to their natural ligands. CRTH2-transfected cells, compared with a Ki of 460 nM for PGJ2 Although eicosanoids were initially considered to be primarily (15). Thus, the binding assay using transfected K562 cells appears proinflammatory mediators, it is becoming clear that their role in ␻ to be more sensitive to structural changes in the side chain of inflammation is quite complex. Both PGD2 and 15d-PGJ2 have PGD2 (with the exception of 13,14-dihydro-15-oxo-PGD2) than proinflammatory effects mediated by the DP2 receptor. PGD2 also biological assays with eosinophils. However, it is important to bear has anti-inflammatory effects via DP1 receptors and can thereby in mind that it is the response of peripheral eosinophils to prosta- inhibit the activation of inflammatory cells, including neutrophils

glandins that is more relevant to the potential in vivo effects of (40) and Langerhans cells (41). However, the DP1 receptor also these compounds. In contrast to its stimulatory effects on eosino- appears to mediate proinﬂammatory effects, given that its deletion ⌬12 phils, -PGJ2 does not stimulate DP1 receptor-mediated activa- resulted in reduced inﬂammation and airway hyperresponsiveness tion of adenylyl cyclase in platelets (37). after Ag challenge of sensitized knockout mice (42). Although

15d-PGD2 is also a potent stimulator of eosinophils, having the 15d-PGJ2 does not activate the DP1 receptor, relatively high con- lowest EC50 (8 nM) for stimulation of CD11b expression of all the centrations of this substance can inhibit macrophages and other compounds tested. Although it has received much less attention cells by both PPAR␥ and non-receptor-mediated mechanisms (4, ⌬12 than the cyclopentenone prostaglandins -PGJ2 and 15d-PGJ2,it 6, 29). Like PGD2, PGE2 also has both pro- and anti-inﬂammatory is a major product of albumin-catalyzed degradation of PGD ,be- effects in various models of inﬂammation (43Ð45). In contrast,

2 Downloaded from ⌬12 ing formed in amounts similar to those of -PGJ2 and consid- most of the eicosanoids formed by the 5-lipoxygenase pathway, erably greater than those of 15d-PGJ2 (32). Therefore, if the de- including leukotriene B4, the cysteinyl-leukotrienes, and 5-oxo- hydration pathway for PGD2 is confirmed to be important ETE, appear to have strictly proinflammatory effects (46), with the physiologically, 15d-PGD2 may be an important proinflammatory exception of the lipoxins, which are highly potent anti- mediator. This compound has been reported to be 100 times less inflammatory agents at nanomolar or subnanomolar concentrations

potent than PGD2 in inhibiting platelet aggregation (38), consistent (47, 48). http://www.jimmunol.org/ with the requirement for a 15-hydroxyl group for activation of the In conclusion, we have shown that 15d-PGJ2 is a potent and DP1 receptor. Thus 15d-PGD2, like 15d-PGJ2, is a selective DP2 selective DP2 receptor agonist that stimulates a variety of re- receptor agonist. sponses in eosinophils, including calcium mobilization, actin po-

We have also examined the effects of other structural modifi- lymerization, and CD11b expression. 15d-PGD2 has similar effects cations of PGD2 on eosinophil activation (Fig. 6). The high po- on CD11b expression. In contrast to PGD2, 15d-PGJ2 has no effect tency of PGD3 (EC50 10 nM) in stimulating CD11b expression is on DP1 receptor-mediated activation of adenylyl cyclase. The consistent with the ability of the DP2 receptor to recognize ago- proinflammatory effects of this compound occur much more rap- nists with substantially modified ␻ side chains. This is consistent idly and at far lower concentrations than its anti-inflammatory ef-

with our previous finding that 13,14-dihydro-15-oxo-PGD2 is a fects on macrophages and other cells, and this should be taken into by guest on October 1, 2021 highly potent DP2 agonist (14). In contrast, modification of the consideration if such compounds are considered for development ⌬5 carboxyl side chain has a much greater effect. Saturation of the as anti-inflammatory agents. Thus 15d-PGJ2 can induce both pro- double bond reduces potency by Ͼ10-fold, whereas reduction of and anti-inflammatory responses, depending on the target cells, its the carboxyl group to a hydroxyl group has a much more substan- concentration, and the time of exposure.

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