Synthetic Prostacyclin Analogs Differentially Regulate Macrophage Function via Distinct Analog-Receptor Binding Specificities

This information is current as David M. Aronoff, Camila M. Peres, Carlos H. Serezani, of October 2, 2021. Megan N. Ballinger, Jennifer K. Carstens, Nicole Coleman, Bethany B. Moore, R. Stokes Peebles, Lucia H. Faccioli and Marc Peters-Golden J Immunol 2007; 178:1628-1634; ; doi: 10.4049/jimmunol.178.3.1628 http://www.jimmunol.org/content/178/3/1628 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Synthetic Prostacyclin Analogs Differentially Regulate Macrophage Function via Distinct Analog-Receptor Binding Specificities1

David M. Aronoff,2,3* Camila M. Peres,2†‡ Carlos H. Serezani,† Megan N. Ballinger,† Jennifer K. Carstens,* Nicole Coleman,* Bethany B. Moore,† R. Stokes Peebles,§ Lucia H. Faccioli,‡ and Marc Peters-Golden†

PGI2 (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI2, in the regulation of both innate and acquired immunity. As PGI2 is unstable, we sought to define the effects of various PGI2 analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, car- baprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were Downloaded from determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in

PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the G␣s protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI2 analogs and more closely resembled the effects of PGE2. Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part

to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies http://www.jimmunol.org/ differences in immunoregulatory properties of clinically administered PGI2 analogs. These studies are the first to explore the capacity of PGI2 to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents. The Journal of Immunology, 2007, 178: 1628–1634.

rostaglandin I2 (prostacyclin; epoprostenol) is an oxygen- In light of its potent vasodilatory properties, PGI2 has been ex- ated metabolite of arachidonic acid formed enzymatically ploited as a pharmacological agent in the treatment of vasocon- P by the sequential activities of cyclooxygenase and PGI strictive/ischemic diseases such as peripheral vascular arterial oc- synthase enzymes (1). It is produced constitutively by vascular clusive disease, cerebrovascular ischemia, and pulmonary arterial by guest on October 2, 2021 endothelial and smooth muscle cells (2) and is induced under in- hypertension (PAH) (1). Although native PGI2 is inherently un-

flammatory conditions in vascular cells (3) and macrophages (4). stable at room temperature with a t1/2 of seconds to minutes (6),

PGI2 is a potent vasodilator and antithrombotic agent (1) whose continuous infusions of a highly alkaline solution (pH 10.2 to 10.8) effects result from binding to a unique heptahelical G protein-cou- are approved by the U.S. Food and Drug Administration (FDA) in pled receptor termed the I prostanoid (IP)4 receptor (5). This re- the treatment of PAH. To combat the technical difficulties associ- ated with administering epoprostenol, more stable synthetic ana- ceptor is G␣s-coupled and activates adenylate cyclase, resulting in an acute burst of intracellular cAMP. logs of PGI2 have been developed and approved for use in PAH. The compound treprostinil (UT-15, Uniprost), which has a half- life of several hours and is stable at room temperature, was ap- proved by the FDA for continuous s.c. infusion. Treprostinil is a *Division of Infectious Diseases and †Division of Pulmonary and Critical Care Med- potent IP receptor agonist (7), although its specificity for this re- icine, Department of Internal Medicine, University of Michigan Health Systems, Ann ceptor is unknown. Iloprost (8) is an inhaled prostacyclin analog Arbor, MI 48109; ‡Departamento de Ana´lises Clı´nicas, Toxicolo´gicas e Bromato- lo´gicas, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o with a half-life between those of epoprostenol and treprostinil that Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil; and §Division of Allergy, Pulmonary, and was also approved by the FDA for use in PAH. Though iloprost is Critical Care Medicine, Vanderbilt University, Nashville, TN 37232 a potent IP receptor agonist that is commonly used in vitro, it also Received for publication September 27, 2006. Accepted for publication November demonstrates significant binding to two of the four E prostanoid 7, 2006. (EP) receptors, namely the G␣q-coupled EP1 and the G␣i-coupled The costs of publication of this article were defrayed in part by the payment of page EP3 subtypes (9). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Cyclic AMP is the quintessential intracellular second messenger

1 This work was supported by the National Institutes of Health Grants AI054660, that amplifies extracellular signals following the binding of G␣s- HL069949, HL078727, HL071586, and HL058897; the American Lung Association coupled receptors by appropriate ligands. Acute increases in Grant RG-8909-N; and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Supe- cAMP have profound anti-inflammatory effects on a number of rior (Capes-Brazil). different cell types involved in both innate and acquired immunity 2 D.M.A. and C.M.P. contributed equally to this work. (10). Our laboratory has studied the effects of cAMP-elevating 3 Address correspondence and reprint requests to Dr. David M. Aronoff, University of Michigan Health System, 5220-D Medical Sciences Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0640. E-mail address: tyl-1-methylxantine; KO, knockout; PAH, pulmonary arterial hypertension; PGI2, [email protected] prostacyclin/epoprostenol; PPAR␤, peroxisome proliferator-activated receptor ␤. 4 Abbreviations used in this paper: IP, G protein-coupled I prostanoid receptor; AM, alveolar macrophage; EP, G protein-coupled E prostanoid receptor; IBMX, 3-isobu- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 1629 compounds on the innate immune system, particularly in the lung Tetrazolium dye reduction assay of bacterial killing (11–13). Recent work demonstrated that PGE2 suppressed critical The ability of bacteria to survive within the macrophages was quantified antimicrobial defense functions of alveolar macrophages (AMs), using a tetrazolium dye reduction assay as described elsewhere (18, 19) and including phagocytosis, bacterial killing, and inflammatory medi- results are expressed as the percentage of survival of phagocytosed ator production, all in a cAMP-dependent manner, mediated via bacteria. the G␣s-coupled EP2 and EP4 receptors (11, 12). Animal models Cytokine measurement in cell culture supernatants have identified important roles for endogenously produced PGE2 in regulating pulmonary host defense (14). Given this ability to Rat or murine macrophages were harvested as described, and the ability of these cells to produce IL-6 and TNF-␣ was assessed by ELISA. Cells were stimulate cAMP production through the IP receptor, we hypothe- treated according to a previously published protocol (13), including a 60- sized that PGI2 analogs might inhibit pulmonary innate immunity min preincubation (37°C with 5% CO2) with compounds of interest in serum-free medium followed by incubation (37°C with 5% CO ) for an in a manner analogous to PGE2. This is particularly relevant given 2 that: 1) PGI analogs are prescribed for PAH; and 2) there are additional 16 h in the presence of LPS 100 ng/ml (or vehicle) and 1% FCS. 2 After this time, cell-free supernatants were collected and analyzed using relatively few studies regarding the effects of PGI2 on innate commercially available ELISA kits for IL-6 (Assay Designs) or TNF-␣ immunity. (R&D Systems) per the manufacturer’s instructions. To test our hypothesis in vitro, we isolated rat AMs and, for Measurement of intracellular cAMP comparison, peritoneal macrophages (PMs) and examined the reg- ulation of phagocytosis, bacterial killing, and inflammatory medi- Macrophages were cultured overnight in 6-well plates in RPMI 1640 at a ϫ 6 ator production by stable PGI analogs. For the first time, the concentration of 3 10 cells/well. Cells were incubated for 30 min with 2 the nonspecific phosphodiesterase inhibitor IBMX (250 ␮M) followed by present investigation identifies differences in the immunoregula-

15 min in the presence or absence of compounds of interest. In some Downloaded from tory properties of clinically administered PGI2 analogs and their experiments the EP2 antagonist AH-6809 (100 ␮M), the EP4 antagonist actions in AMs and PMs. These findings may predict important ONO-AE3-208 (10 ␮M), or vehicle (DMSO) was added with IBMX for 30 differences regarding the risk of infection for patients receiving min before adding PGI2 analogs. The doses of AH-6809 and ONO-AE3- such agents. 208 were based on previous reports (11, 20). Culture supernatants were aspirated and the cells were lysed by incubation for 20 min with 0.1 M HCl (22°C), followed by disruption using a cell scraper. Intracellular cAMP Materials and Methods levels were determined by ELISA according to the manufacturer (Assay Animals Designs). http://www.jimmunol.org/ Pathogen-free 125- to 150-g female Wistar rats were obtained from Charles Immunoblot analysis River Laboratories. Mice with a targeted disruption of the EP2 gene (15) Western blot analysis was performed as previously described (21). Briefly, backcrossed over 10 generations onto a C57BL/6 background (designated whole cell protein extracts were obtained by lysing freshly harvested AMs as EP2 knockout (KO) mice) were a gift from S. Narumiya (Kyoto Uni- and PMs in a buffer (50 mM Tris-HCl (pH 7.4), 25 mM KCl, 5 mM MgCl2, versity, Kyoto, Japan) and were obtained from ONO Pharmaceutical and and 0.2% Nonidet P-40) supplemented with protease inhibitors (Roche bred in the University of Michigan Unit for Laboratory Animal Medicine Diagnostics). Protein samples (40 ␮g) were resolved on 10% Tris-HCl (Ann Arbor, MI). Wild-type C57BL/6 mice were purchased from The polyacrylamide gels and subsequently transferred to a nitrocellulose mem- Jackson Laboratory. Animals were treated according to National Institutes brane. Membranes were probed with commercially available rabbit poly- of Health guidelines for the use of experimental animals with the approval clonal EP2 and IP receptor Abs (Cayman Chemicals) or a mouse mono- by guest on October 2, 2021 of the University of Michigan Committee for the Use and Care of Animals. clonal anti-␤-actin Ab (Sigma-Aldrich) followed by HRP-conjugated anti- Reagents rabbit or anti-mouse secondary Abs and ECL Plus detection reagents (Amersham Biosciences). Relative band densities were determined by den- RPMI 1640 cell culture medium and a penicillin/streptomycin/amphoteri- sitometric analysis using National Institutes of Health Image software and cin B solution were purchased from Invitrogen Life Technologies. Tryptic the ratios were calculated. The results were expressed as the ratio of IP to soy broth was supplied by Difco. LPS of Escherichia coli strain 055:B5, that of ␤-actin. In all instances, density values of bands were corrected by cytochalasin D, SDS, o-phenylenediamine dihydrochloride, saponin, MTT, subtraction of the background values. and peroxidase-labeled monoclonal anti-rabbit IgG were purchased from Sigma-Aldrich. The nonspecific phosphodiesterase inhibitor 3-isobutyl-1- Statistical analysis methylxantine (IBMX) was purchased from EMD Biosciences. PGE2, car- Data are represented as mean Ϯ SEM and were analyzed with the Prism 4.0 baprostacyclin, iloprost, treprostinil, AH-6809 (EP2 receptor antagonist), statistical program (GraphPad). Comparisons among three or more exper- and rabbit polyclonal anti-IP and anti-EP2 receptor Abs were from Cayman imental groups were performed with ANOVA followed by the Bonferroni Chemicals. ONO-AE3-208 (EP4 receptor antagonist) was provided by correction as indicated. Differences were considered significant if p Յ 0.05. ONO Pharmaceutical. Compounds requiring reconstitution were dissolved All experiments were performed on at least three separate occasions unless in DMSO. Required dilutions of all compounds were prepared immediately otherwise specified. before use, and equivalent quantities of vehicle were added to the appro- priate controls. Results ␥ Isolation and culture of alveolar and peritoneal macrophages PGI2 analogs differentially regulate Fc R-mediated phagocytosis in rat macrophages Resident AMs from rats and mice and PMs from rats were obtained via lung and peritoneal lavages. respectively, as previously described (16, 17) Iloprost and treprostinil are approved for use in the treatment of and resuspended in RPMI 1640 to a final concentration of 1–4 ϫ 106 PAH. The former is a standard and well-characterized IP receptor cells/ml. Cells were allowed to adhere to tissue culture-treated slides or agonist that also binds EP1 and EP3 receptors (9), whereas trepro- plates for 1 h (37°C with 5% CO2) followed by two washes with warm RPMI 1640. Cells were cultured overnight in RPMI 1640 containing 10% stinil has an extended half-life and chemical stability but its spec- FBS and 1% penicillin/streptomycin/amphotericin B before use. The fol- ificity for the IP receptor is unclear. We assessed the ability of lowing day cells were washed two times with warm medium to remove iloprost and treprostinil to inhibit FcR-mediated phagocytosis, a nonadherent cells. process known to be inhibited by acute rises in intracellular cAMP Fc␥R-mediated phagocytosis assays (11). Native PGI2 (epoprostenol) was not used due to its inherent instability (22). We compared the potencies of these synthetic The phagocytosis of IgG-opsonized, nonviable, FITC-labeled Escherichia PGI analogs with that of PGE , which suppresses FcR phagocy- coli BioParticles (Molecular Probes) was assessed as previously reported 2 2 (11). Drugs of interest were added 5 min before phagocytosis was initiated. tosis through cAMP-mediated signaling (11). Rat AMs or PMs Results are expressed as a percentage of the control, to which only vehicle were pretreated for 5 min with iloprost, treprostinil, or PGE2 be- was added. fore incubation with IgG-opsonized E. coli. A concentration of 1 1630 PROSTACYCLIN ANALOGS MODULATE INNATE IMMUNITY

␮M of these compounds was used for interdrug comparisons be- IL-6 production following LPS treatment in both AMs and PMs. ␮ cause: 1) 1 M PGE2 predictably evokes a significant increase in Although this was indeed the case for iloprost and carbaprostacy- cAMP and substantially inhibits FcR-mediated phagocytosis; and clin in PMs, as was also observed for PGE2, neither iloprost nor 2) 1 ␮M iloprost elicited near maximal cAMP response in rat carbaprostacyclin (1 ␮M) affected LPS-stimulated IL-6 release by macrophages (see Fig. 4). AMs (Fig. 2A). By contrast, treprostinil significantly up-regulated Interestingly, iloprost inhibited the FcR-mediated phagocytosis LPS-stimulated IL-6 in both AMs and PMs (Fig. 2B). These data of IgG-E. coli by 51.4 Ϯ 6.5% in PMs but did not significantly are in accord with our findings in phagocytosis and indicate that

impair phagocytosis in AMs (Fig. 1A). Dose-response experiments treprostinil behaves more like PGE2 in AMs than either of the revealed that iloprost significantly inhibited phagocytosis in PMs other two PGI2 analogs. more potently than in AMs across all drug concentrations exam- ined ( p Ͻ 0.001, comparing suppression in PMs vs AMs for each PGI2 analogs differentially suppress bacterial killing by rat dose; Fig. 1B). Similar results were seen with carbaprostacyclin, macrophages another synthetic IP receptor agonist, which is not FDA approved Along with phagocytosis and the generation of inflammatory me- for use in humans but is commonly used in vitro (not shown). diators, microbial killing is a critical defense function of the mac-

As we have previously shown in AMs (10), PGE2 also inhibits rophage. Little is known about the direct effects of PGI2 or its FcR-mediated phagocytosis by PMs (Fig. 1A). We anticipated that analogs on the regulation of leukocyte bactericidal activity (25).

treprostinil would show a similar profile as iloprost and carbapros- We therefore examined the effect of stable PGI2 analogs on bac- tacyclin; however, treprostinil (1 ␮M) inhibited FcR-mediated terial killing using the Gram-negative pathogen Klebsiella pneu- Ϯ phagocytosis effectively in both AMs and PMs by 35.0 5.6 and moniae (26). As demonstrated in Fig. 3, both PGE2 and treprostinil Downloaded from 58.5 Ϯ 5.7%, respectively (Fig. 1A), an effect parallel to that of dramatically increased the ability of phagocytosed K. pneumoniae

PGE2. Results similar to these were obtained using an alternative to survive within both AMs and PMs. By contrast, both iloprost assay for FcR-mediated phagocytosis with IgG-coated sheep and carbaprostacyclin were relatively weak at inhibiting killing in RBCs (data not shown). either cell type. The ability of carbaprostacyclin to block bacteri- cidal mechanisms was minimal and only reached statistical signif- PGI2 analogs differentially regulate LPS-stimulated IL-6 icance in the PM (Fig. 3). production in rat macrophages http://www.jimmunol.org/

Depending on the cell of interest and the experimental approach, cAMP elevation by PGI2 analogs reflects their acute increases in cAMP can be found to either enhance or sup- immunomodulatory properties

press LPS-stimulated IL-6 production (23, 24). In rat AMs and The IP receptor predominantly signals through a G␣s-coupled ac- PMs, however, cAMP augments LPS-stimulated IL-6 generation tivation of adenylate cyclase. We have previously demonstrated

(13). We therefore hypothesized that PGI2 analogs would increase that immunosuppression of macrophage functions by PGE2 fol-

lows a similar G␣s-coupled receptor-based mechanism (11). As by guest on October 2, 2021

FIGURE 1. Prostacyclin analogs differentially inhibit FcR phagocytosis in rat macrophages. A, Rat AMs and PMs were harvested as described in FIGURE 2. Prostacyclin analogs differentially enhance IL-6 production Materials and Methods. Cells were pretreated with iloprost, treprostinil by LPS-treated macrophages. Rat AMs or PMs were preincubated for 60 ␮ ␮ (TRE), or PGE2 (each at 1 M) or vehicle control for 5 min and then min (37°C with 5% CO2) with PGE2, iloprost, or carbaprostacyclin (1 M) ␮ challenged with E. coli opsonized with specific rabbit polyclonal IgG. (A) and PGE2 or treprostinil (1 M) (B) in serum-free medium followed by Phagocytic indexes were calculated as described (11) and expressed as a incubation for an additional 16 h in the presence of LPS 100 ng/ml (or percentage of the control value to which no drugs were added. B, Cells vehicle) and 1% FCS. After this time, cell-free supernatants were collected were preincubated for 5 min with iloprost (1–10 ␮M) and phagocytosis and analyzed for IL-6 using a commercially available ELISA kit. Data are ,ءءء .p Ͻ 0.001 vs control. expressed as increase in nanograms per milliliter above LPS alone ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .was assessed as in A Mean (Ϯ SEM) data are shown from three different experiments each per- p Ͻ 0.001. Data are mean (Ϯ SEM) from triplicate wells of one experiment formed in sextuplet. representative of three. The Journal of Immunology 1631

cAMP levels increased 23-fold in PMs but only 3.9-fold in AMs. ␮ Both treprostinil and PGE2 (1 M) increased cAMP to similar levels in AMs and PMs (Fig. 4C).

PMs express more IP receptor than AMs The fact that iloprost and carbaprostacyclin increase cAMP and suppress the effector functions in PMs to a greater degree than in AMs suggested that AMs and PMs may differ in their levels of expression of the IP receptor. Protein preparations from lysates of freshly harvested, unstimulated rat AMs and PMs were subjected to Western blot analysis for the detection of IP receptor protein FIGURE 3. Prostacyclin analogs differentially inhibit bacterial killing using a polyclonal Ab (Fig. 5). In view of the similar suppressive by rat macrophages. Rat AMs or PMs were infected with 50:1 opsonized effect of PGE2 in both cell populations, we also examined EP2 K. pneumoniae. Thirty minutes after infection, the cells were incubated expression. We did not explore differences in the other G␣ -cou- with PGE , treprostinil (TRE), iloprost, or carbaprostacyclin (1 ␮M) or s 2 pled EP receptor, EP4, as we have previously demonstrated that rat vehicle control. Microbicidal activity was assessed colorimetrically as de- AMs express very little functional EP4 (11). Although the EP2 scribed in Materials and Methods. Data are expressed as the mean Ϯ SEM percentage of survival of ingested bacteria from three independent exper- receptor was expressed at similar levels in both AMs and PMs, we -p Ͻ 0.001 compared consistently found that the IP receptor was present in greater abun ,ءء ;p Ͻ 0.05 ,ء .iments, each performed in triplicate with control. dance (ϳ2.3-fold) in the latter population (Fig. 5). Thus, differ- Downloaded from ences in cAMP generation (and downstream immunoregulatory pathways) between AMs and PMs in response to iloprost and car- shown in Fig. 4A, treating AMs for 15 min with either iloprost or baprostacyclin correlate with differences in IP receptor expression. carbaprostacyclin (1 ␮M) failed to enhance cAMP significantly in Treprostinil activates the EP2 receptor AMs, in stark contrast to both treprostinil and PGE2. It is unlikely that we missed an early and transient increase in cAMP generated The above data demonstrate that treprostinil is a more potent in- by iloprost or carbaprostacyclin, as these experiments were con- http://www.jimmunol.org/ hibitor of AM antimicrobial defenses than other PGI2 analogs, a ducted in the presence of the PDE inhibitor IBMX, which sustains phenomenon reflected in its ability to increase cAMP in AMs more cAMP elevations. By contrast with these results in AMs, both potently than either iloprost or carbaprostacyclin. This might re- iloprost (Fig. 4B) and carbaprostacyclin (not shown) significantly flect the ability of treprostinil to stimulate the relatively low-abun- increased cAMP in PMs. At a concentration of 1 ␮M iloprost, dance IP receptor more potently than other agonists (7). However, as we were unable to find published data regarding the specificity of treprostinil, we hypothesized that it might bind not only the IP receptor but also the EP2 receptor, the latter being the key receptor

mediating cAMP responses to PGE2 in the rat AM (11). To test by guest on October 2, 2021 this hypothesis, we pretreated rat AMs for 30 min with antagonists for either the EP2 receptor (AH-6809; 100 ␮M) or the EP4 recep- tor (ONO-AE3-208; 10 ␮M) followed by a 15-min treatment with 1 ␮M treprostinil (Fig. 6A). As illustrated, AH-6809 blocked ϳ74% of the increase in cAMP stimulated by treprostinil, whereas the EP4 selective antagonist had no significant effect. We next examined the ability of treprostinil to stimulate cAMP in AMs harvested from EP2 KO mice and compared their responses to cells from wild-type animals (Fig. 6B). Confirming our pharma- cological data, the absence of the EP2 receptor reduced the trepro- stinil-stimulated increase in cAMP by ϳ68%. To document that the lack of an EP2 receptor could reduce the potency of treprostinil

FIGURE 4. Effect of prostacyclin analogs and PGE2 on intracellular cAMP concentration ([cAMP]) in rat macrophages. Rat AMs (A–C)or PMs (B and C) were pretreated for 30 min with IBMX (250 ␮M) or ve-

hicle, followed by iloprost, treprostinil (TRE), or PGE2 at a final concen- tration of 1 ␮M (or as indicated in B) for 15 min. Intracellular cAMP FIGURE 5. Rat PMs express higher levels of IP receptors than do AMs. concentrations were determined as described in Materials and Methods. Whole cell lysates from PMs and AMs were prepared and Western blot Data are expressed as relative units from the control value (A and B)to analysis was performed using rabbit polyclonal anti-IP and anti-EP recep- tor Abs or mouse monoclonal anti-␤-actin Abs as described in Materials ,ءء ;p Ͻ 0.05 ,ء .(which only vehicle was added or as absolute values (C p Ͻ 0.001 compared with control. Data from A are mean Ϯ SEM from and Methods. One of two representative Western blots yielding similar three to five separate experiments, and representative experiments are results is shown. Band densitometry values expressing the density ratio of shown in B and C (n ϭ 3). IP receptor expression to ␤-actin are shown. 1632 PROSTACYCLIN ANALOGS MODULATE INNATE IMMUNITY

munity (31). However, data regarding the influence of PGI2 on lung innate immunity are lacking. Such investigations are impor-

tant given that stable, long-acting synthetic analogs of PGI2 are in clinical use in patients with chronic pulmonary diseases (e.g., PAH) whose lower respiratory tract defense mechanisms may be impaired. Our previous results have demonstrated that the suppressive ef-

fects of PGE2 on AM functions follow its ability to acutely elevate cAMP via G␣s-coupled receptors (11, 12). We therefore postulated that PGI2, which itself signals via the G␣s-coupled IP receptor, would suppress innate immunity in a manner similar to that of

PGE2. Surprisingly, our studies found that standard PGI2 analogs (iloprost and carbaprostacyclin) only weakly regulated AM phago- cytosis, inflammatory mediatory generation, and bacterial killing

but were as potent as PGE2 in regulating PMs. For these studies we examined FcR-mediated phagocytosis, a standard model of phago- cytosis about which much is understood regarding its regulation by eicosanoids and cAMP (11, 12). We were unable to identify prior

studies of PGI2-regulation of FcR-mediated phagocytosis and now demonstrate that IP receptor activation inhibits the ingestion of Downloaded from IgG-opsonized targets in resident rat PMs to a much greater extent than in AMs. That the inhibitory effects of IP receptor activation might extend beyond FcR-mediated uptake is suggested by a re-

port that PGI2 was able to block the fibronectin-mediated ingestion of gelatinized sheep RBCs by casein-elicited rat PMs (32).

FIGURE 6. The EP2 receptor mediates some of the effects of trepros- http://www.jimmunol.org/ To assess whether the effect of iloprost and carbaprostacyclin tinil (TRE). A, Rat AMs were preincubated for 30 min with the EP2 an- tagonist AH-6809 (100 ␮M), the EP4 antagonist ONO-AE3-208, or vehi- was limited to phagocytosis, we turned our attention to the ability cle, followed by treprostinil at a final concentration of 1 ␮M for 15 min. of these drugs to regulate LPS-stimulated IL-6. IL-6 is a cytokine IBMX was used to inhibit phosphodiesterase degradation of cAMP. B, with mixed pro and anti-inflammatory effects in the lung (33). We Murine AMs from wild-type (EP2ϩ/ϩ) or EP2-null (EP2Ϫ/Ϫ) mice were chose this cytokine for a number of reasons. First, IL-6 is well ␣ treated with treprostinil as in panel A. C, Mouse TNF- production was recognized to be regulated by cAMP and, in particular, both PGE2 measured in the cell-free supernatants from EP2ϩ/ϩ or EP2Ϫ/Ϫ AMs and PGI2 have been shown to modulate its synthesis during LPS treated with LPS overnight following a 1-h preincubation with either stimulation (34, 35). Second, in the rat model both AM and PM Ͻ ءءء ␮ ␮ treprostinil (1 M) or PGE2 (1 M). , p 0.001. IL-6 synthesis are profoundly regulated by PGE under conditions 2 by guest on October 2, 2021 of LPS treatment (13). Lastly, as discussed below, IL-6 production by human AMs has been shown to be regulated by treprostinil in regulating inflammatory functions of the AM, we pretreated during LPS exposure (29). Our data (Fig. 2) demonstrating that

EP2-null or wild-type AMs for 1 h with either treprostinil or PGE2 neither iloprost nor carbaprostacyclin stimulate IL-6 production in followed by an overnight incubation with LPS (100 ng/ml). As rat AMs but do in PMs suggest that there is a proximal signaling shown (Fig. 6C), the ability of treprostinil to suppress TNF-␣ pro- difference between AMs and PMs that explains the differing sus- duction was reduced in the absence of the EP2 receptor to the same ceptibility of various functions of these cells to these PGI2 analogs. extent as was the activity of PGE2. It is notable that the lack of EP2 The killing of K. pneumoniae by AMs can be inhibited by ␣ did not completely abrogate the ability of PGE2 to decrease TNF- PGE2-induced increases in cAMP (12). Regulation of bacterial production (Fig. 6C). This most likely reflects the fact that AMs killing by PGI2 has only been studied directly in neutrophils, from EP2 KO animals express increased amounts of the G␣s-cou- where it was not found to have an effect (25). We find in both AMs pled EP4 receptor, perhaps to compensate for the lack of EP2 (14). and PMs (Fig. 3) that iloprost and carbaprostacyclin are weak in-

hibitors of bacterial killing when compared with PGE2. The reason Discussion that these compounds are so much weaker than PGE2 in suppress-

PGI2 is a cyclooxygenase-derived lipid mediator well known for ing bacterial killing by PMs is not entirely clear and requires fur- its regulatory effects on vascular endothelial and smooth muscle ther study. However, these experiments again revealed differential cells as well as platelets. Less appreciated, however, is the influ- regulation of AMs and PMs by these PGI2 mimics, as both com- ence PGI2 has on the immune system. PGI2 has been postulated to pounds were significantly more potent at inhibiting bacterial kill- play a key role in regulating both innate and acquired immunity ing in the latter cell. Whether such effects are relevant in vivo has and the effects are, for the most part, immunosuppressive or anti- yet to be determined. inflammatory, resulting from IP-mediated increases in cAMP (re- Because the IP receptor is G␣s coupled, we were able to measure viewed in Ref. 27). Inhibitory effects on innate immunity include intracellular cAMP responses as a surrogate marker of IP activa- the suppression of adhesion molecule expression by endothelial tion in the presence of iloprost and carbaprostacyclin (Fig. 4). cells and leukocytes (27), the reduction of reactive oxygen inter- These data not only confirmed differences in sensitivity of AMs mediate production by neutrophils (28), the impairment of inflam- and PMs to these drugs but importantly suggested a mechanism matory cytokine generation by pathogen-stimulated macrophages underlying such sensitivities: namely, that the IP receptor itself (29) and dendritic cells (R.S. Peebles, unpublished observation), might be differentially expressed by these cells. Indeed, by West- and the inhibition of NK-cell mediated cytotoxicity (30). The over- ern blot analysis we found greater IP receptor protein expression in production of PGI2 is believed to play a role in the pathogenesis of rat PMs than in AMs. Whether such discrepant receptor expression certain bacterial infections, perhaps by direct effects on innate im- is the sole explanation for differences in cAMP synthesis between The Journal of Immunology 1633

the two macrophage cell types requires further study. It remains disease-related factors that might influence the risks posed by PGI2 possible that the coupling between the IP receptor and adenylate analogs in particular subpopulations of patients. cyclases or the types and amounts of adenylate cyclase isoforms expressed in the two cells may differ. It is unlikely that differences Disclosures in phosphodiesterase isoform expression explains our results, as We have no financial conflict of interest. we inhibited all cAMP phosphodiesterases with IBMX. A quite unexpected result was the finding that treprostinil did References not share the same regulatory phenotype as the other PGI2 analogs. 1. Vane, J., and R. E. Corin. 2003. Prostacyclin: a vascular mediator. Eur. J. Vasc. Treprostinil is clearly a potent IP receptor agonist (7), but its spec- Endovasc. Surg. 26: 571–578. ificity for that receptor has not been published. We have recently 2. Wu, K. K., and J. Y. Liou. 2005. Cellular and molecular biology of prostacyclin synthase. Biochem. Biophys. Res. Commun. 338: 45–52. found that treprostinil modulates T lymphocyte and dendritic cell 3. Wen, F. Q., K. Watanabe, H. Tanaka, and M. Yoshida. 1997. Cytokines and functions in an IP receptor-independent fashion (R.S. Peebles, un- lipopolysaccharide enhance basal and thrombin-stimulated production of PGI2 by published observation). Such data suggest that this drug may bind cultured human pulmonary artery smooth muscle cells. Prostaglandins Leukot. Essent. Fatty Acids. 56: 185–192. other G␣s-coupled receptors and we hypothesized that the EP2 4. Brock, T. G., R. W. McNish, and M. Peters-Golden. 1999. Arachidonic acid is receptor might be one such unintended target. Although treprosti- preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglan- din E2. J. Biol. Chem. 274: 11660–11666. nil has been suggested to also be a ligand for the peroxisome 5. Hata, A. N., and R. M. Breyer. 2004. Pharmacology and signaling of prostaglan- proliferator-activated receptor ␤ (PPAR␤) receptor (36), we did din receptors: multiple roles in inflammation and immune modulation. Pharma- not consider this to be a likely mechanism to explain our findings. col. Ther. 103: 147–166. Our treatments were brief (minutes) in the studies of cAMP ele- 6. Cho, M. J., and M. A. Allen. 1978. Chemical stability of prostacyclin (PGI2)in

aqueous solutions. Prostaglandins 15: 943–954. Downloaded from vation, FcR-mediated phagocytosis, and bacterial killing, whereas 7. Clapp, L. H., P. Finney, S. Turcato, S. Tran, L. J. Rubin, and A. Tinker. 2002. PPAR␤ is a transcription factor whose effects would be expected Differential effects of stable prostacyclin analogs on smooth muscle proliferation and cyclic AMP generation in human pulmonary artery. Am. J. Respir. Cell Mol. to require a number of hours. Though it remains possible that the Biol. 26: 194–201. effect of treprostinil on cytokine synthesis involved the PPAR␤ 8. Olschewski, H., D. Walmrath, R. Schermuly, A. Ghofrani, F. Grimminger, and system because those experiments were conducted in the presence W. Seeger. 1996. Aerosolized prostacyclin and iloprost in severe pulmonary hy- pertension. Ann. Intern. Med. 124: 820–824. of treprostinil for Ͼ16 h, we have previously shown that cAMP 9. Abramovitz, M., M. Adam, Y. Boie, M. Carriere, D. Denis, C. 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