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Prostaglandin E2 Inhibits Alveolar Macrophage Phagocytosis through an E-Prostanoid 2 Receptor-Mediated Increase in Intracellular Cyclic AMP This information is current as of September 25, 2021. David M. Aronoff, Claudio Canetti and Marc Peters-Golden J Immunol 2004; 173:559-565; ; doi: 10.4049/jimmunol.173.1.559 http://www.jimmunol.org/content/173/1/559 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Prostaglandin E2 Inhibits Alveolar Macrophage Phagocytosis through an E-Prostanoid 2 Receptor-Mediated Increase in Intracellular Cyclic AMP1,2

David M. Aronoff,*† Claudio Canetti,† and Marc Peters-Golden3†

Prostaglandin E2 is a potent lipid mediator of inﬂammation that effects changes in cell functions through ligation of four distinct

G protein-coupled receptors (E-prostanoid (EP)1, EP2, EP3, and EP4). During pneumonia, PGE2 production is enhanced. In the ␥ present study, we sought to assess the effect of endogenously produced and exogenously added PGE2 on FcR -mediated phagocytosis of bacterial pathogens by alveolar macrophages (AMs), which are critical participants in lung innate immunity. We also sought to characterize the EP receptor signaling pathways responsible for these effects. PGE2 (1–1000 nM) dose-dependently suppressed the phagocytosis by rat AMs of IgG-opsonized erythrocytes, immune serum-opsonized Klebsiella pneumoniae, and Downloaded from IgG-opsonized Escherichia coli. Conversely, phagocytosis was stimulated by pretreatment with the cyclooxygenase inhibitor indomethacin. PGE2 suppression of phagocytosis was associated with enhanced intracellular cAMP production. Experiments using both forskolin (adenylate cyclase activator) and rolipram (phosphodiesterase IV inhibitor) conﬁrmed the inhibitory effect of cAMP stimulation. Immunoblot analysis of rat AMs identiﬁed expression of only EP2 and EP3 receptors. The selective EP2 agonist butaprost, but neither the EP1/EP3 agonist sulprostone nor the EP4-selective agonist ONO-AE1-329, mimicked the effects of PGE2

on phagocytosis and cAMP stimulation. Additionally, the EP2 antagonist AH-6809 abrogated the inhibitory effects of both PGE2 http://www.jimmunol.org/ and butaprost. We confirmed the specificity of our results by showing that AMs from EP2-deficient mice were resistant to the inhibitory effects of PGE2. Our data support a negative regulatory role for PGE2 on the antimicrobial activity of AMs, which has important implications for future efforts to prevent and treat bacterial pneumonia. The Journal of Immunology, 2004, 173: 559–565.

neumonia is the leading cause of death from infection in modulators of innate immunity (4, 5). PGE2 in particular has been the United States (1) and its global mortality is ϳ4.3 mil- shown to modulate immune and inﬂammatory responses (5, 6). It lion victims per year (2). This problem is compounded by is a product of the cyclooxygenase (COX) cascade, which includes

P by guest on September 25, 2021 growing numbers of immunosuppressed patients and multidrug- two distinct isoforms of COX, the constitutive COX-1 and the resistant microorganisms. Developing more effective agents for the (usually) inducible COX-2, as well as constitutive and inducible prevention and treatment of pneumonia requires a better under- PGE synthase enzymes. Production of PGE2 is enhanced at sites of standing of how innate pulmonary defense mechanisms are regu- inflammation, in which it demonstrates both pro- and anti- 4 lated. In the lung periphery the alveolar macrophage (AM) is the inflammatory effects. resident defender of mucosal sterility, patrolling the alveolar epi- PGE2 promotes inflammation through endothelial cell-mediated thelial surface and clearing organisms by phagocytosis and intra- vasodilatation (producing warmth, erythema, and edema), facili- cellular killing (3). The antimicrobial activities of this sentinel of tating the recruitment of circulating leukocytes to areas of infec- innate immunity are regulated by a number of autocrine and para- tion (7). In addition, PGE enhances leukocyte production of the crine factors including cytokines, chemokines, and lipids. 2 inflammatory cytokine IL-6 (8). It is less well appreciated that Lipid metabolites of arachidonic acid (AA), including PGs and PGE has potent immunosuppressive properties, including the di- leukotrienes, have emerged as potent endogenous mediators and 2 rect inhibition of leukocyte chemotaxis (9), reactive oxygen inter- mediate production (10), AA release (11), leukotriene synthesis Divisions of *Infectious Diseases and †Pulmonary and Critical Care Medicine, De- (12), and the generation of myriad proinflammatory cytokines partment of Internal Medicine, University of Michigan Health System, Ann Arbor, MI (13). By contrast, PGE enhances the production of the immuno- 48109-0642 2 suppressive cytokine IL-10 (14). Received for publication January 16, 2004. Accepted for publication April 27, 2004. Of interest, PGE production is enhanced in the lung during The costs of publication of this article were defrayed in part by the payment of page 2 charges. This article must therefore be hereby marked advertisement in accordance bacterial pneumonia (15) and overproduction of PGE2 has also with 18 U.S.C. Section 1734 solely to indicate this fact. been described in a number of conditions characterized by an in- 1 This work was supported by the National Institutes of Health Grants HL007749 and creased susceptibility to infection, including cancer, aging, HIV HL058897, and Conselho Nacional de Pesquisa (CNPq-Brazil). infection, and malnutrition (16Ð19). That the net effect of PGE2 is 2 Presented in part at the 41st Annual Meeting of the Infectious Diseases Society of suppressive in the context of infection is supported by studies dem- America (Abstract 715), San Diego, CA, October 2003. onstrating that administration of a COX inhibitor enhanced micro- 3 Address correspondence and reprint requests to Dr. Marc Peters-Golden, 6303 Med- ical Science Research Building III, Box 0642, 1150 West Medical Center Drive, bial clearance and survival in animal models of infection (20, 21). University of Michigan Health System, Ann Arbor, MI 48109-0642. E-mail address: PGE2 effects changes in target cells via signaling through four [email protected] distinct cell membrane-associated G protein-coupled E-prostanoid 4 Abbreviations used in this paper: AM, alveolar macrophage; AA, arachidonic acid; RFU, relative fluorescence unit; COX, cyclooxygenase; EP, E-prostanoid; PI, phago- (EP) receptors, termed EP1, EP2, EP3, and EP4 (22). EP1 receptor 2ϩ cytic index. activation provokes Gq-coupled increases in intracellular Ca ,

␮ EP2 and EP4 receptors signal predominantly through Gs, increas- inhibitor cytochalasin D (5 g/ml) (33). A minimum of four replicate wells per condition was studied in each experiment. ing cAMP, and the EP3 receptor most often reduces cAMP via Gi coupling after PGE2 ligation (22). PGE2 has been shown to either stimulate or inhibit phagocytosis by macrophages (23Ð29). Noth- Fluorometric phagocytosis assay with FITC-labeled Escherichia ing is known about AM expression of EP receptors or the effects coli of PGE2 on bacterial ingestion by these cells. In the present study, Paraformaldehyde-inactivated, FITC-labeled E. coli BioParticles (3 ϫ 108 we provide evidence that endogenous and exogenous PGE2 inhibit E. coli per milligram of solid; Molecular Probes, Eugene, OR) were sus- FcR␥-mediated phagocytosis by AMs via EP2-mediated cAMP pended in PBS at a concentration of 20 mg/ml followed by brief sonication signaling. on ice. E. coli were opsonized with specific rabbit polyclonal IgG (Mo- lecular Probes) according to the manufacturer’s directions. Opsonized E. coli (IgG-E. coli) was further diluted in DMEM for use in phagocytosis Materials and Methods experiments. Rat AMs were cultured overnight in 96-well tissue culture- Animals treated dishes (Costar, Corning, NY) at a density of 4 ϫ 105 cells/well. Murine AMs were cultured in a similar fashion in 384-well plates (Costar) Mice with a targeted disruption of the EP2 gene (30) backcrossed over 10 at a density of 5 ϫ 104 per well. Cells were then washed two times with generations onto a C57BL/6 background (designated as EP2 knockout warm DMEM and preincubated with compounds of interest, followed by mice) were obtained from ONO Pharmaceutical (Osaka, Japan) and bred in the application of IgG-E. coli (MOI ϭ 30:1). After 30 min incubation in the the University of Michigan Unit for Laboratory Animal Medicine (Ann dark (37¡C, 5% CO2), uningested bacteria were washed away with PBS Arbor, MI). Wild-type C57BL/6 mice were purchased from The Jackson and residual extracellular FITC (representing cell-adherent IgG-E. coli) Laboratory (Bar Harbor, ME). Pathogen-free 125Ð150 g female Wistar rats was quenched with trypan blue (250 ␮g/ml; Molecular Probes) for 1 min. (Charles River Breeding Laboratories, Portage, MI) were also used. Ani- Fluorescence was determined with a microplate fluorometer (485 /535 , ex em Downloaded from mals were treated according to National Institutes of Health guidelines for SPECTRAFluor Plus; Tecan, Research Triangle Park, NC). Independent the use of experimental animals with the approval of the University of experiments were performed in quadruplicate. Fluorometric data were ex- Michigan Committee for the Use and Care of Animals. pressed in arbitrary relative fluorescence units (RFU) and corrected for background fluorescence using wells containing only AMs and medium. Reagents The PI represented the fluorescence emitted from intracellular (phagocy- DMEM without phenol red, RPMI 1640, and penicillin/streptomycin/am- tosed) bacteria (RFUi) and was determined by subtracting the fluorescence photericin B solution were purchased from Life Technologies-Invitrogen from unquenched, extracellular bacteria (RFUex) from the total fluores- (Carlsbad, CA). Tryptic soy broth was supplied by Difco (Detroit, MI). cence of the experimental well (RFUtotal). The RFUex was determined us- http://www.jimmunol.org/ Aspirin, cytochalasin D, ibuprofen, indomethacin, o-phenylenediamine di- ing the fluorescence of cells exposed for 45 min to the phagocytosis in- ␮ ϭ ϭ hydrochloride, and SDS were from Sigma-Aldrich (St. Louis, MO). AH- hibitor cytochalasin D (5 g/ml) (34). Thus, the PI RFUi Ϫ RFUtotal RFUex. The ability of cytochalasin D to completely inhibit 6809, butaprost free acid, PGE2, sulprostone, and U-46619 were from Cay- man Chemicals (Ann Arbor, MI). ONO-AE1-329 was a generous gift from phagocytosis was confirmed by fluorescent microscopy. ONO Pharmaceutical. Forskolin and rolipram were purchased from Cal- biochem (San Diego, CA). Compounds requiring reconstitution were dis- Microcolorimetric erythrocyte phagocytosis assay solved in either ethanol or DMSO. Required dilutions of all compounds were prepared immediately before use, and equivalent quantities of vehicle The phagocytosis of sheep RBCs (sRBCs) by rat AMs was assessed as were added to the appropriate controls. previously described (35). Briefly, AMs were plated and cultured overnight ϫ 5 in 96-well culture-treated dishes (BD Biosciences) at a density of 2 10 by guest on September 25, 2021 Cell isolation and culture cells/well. sRBCs (ICN Pharmaceuticals, Costa Mesa, CA) were opsonized with a subagglutinating concentration of polyclonal, rabbit anti-sRBC IgG Resident AMs from mice and rats were obtained via ex vivo lung lavage as (Cappel Organon Teknika, Durham, NC) as previously described (36). previously described (31) and resuspended in RPMI 1640 to a final con- AMs were then washed twice with warm DMEM and preincubated with centration of 1Ð4 ϫ 106 cells/ml. Cells were allowed to adhere to tissue ␮ ␮ cytochalasin D (5 g/ml, 45 min), indomethacin (10 M, 30 min), or PGE2 culture-treated slides or plates for1h(37¡C, 5% CO2) followed by two (1Ð1000 nM, 5 min). Following preincubation, opsonized sRBCs were Ͼ washes with warm RPMI 1640, resulting in 99% of adherent cells iden- added at a ratio of 50:1 (sRBC to AMs) and cultures were incubated for an tified as AMs by use of a modified Wright-Giemsa stain (Diff-Quik; Amer- additional 90 min at 37¡C. Wells were then washed three times with PBS ican Scientific Products, McGraw Park, IL) (31). Cells were cultured over- to remove noningested erythrocytes and 100 ␮l of 0.3% SDS in PBS were night in RPMI 1640 containing 10% FBS and 1% penicillin/streptomycin/ added to each well for 10 min. Serial dilutions of known amounts of sRBCs amphotericin B before use. The following day cells were washed two times were added to separate wells before the addition of the SDS solution to with warm medium to remove nonadherent cells. derive a standard curve. Lastly, 100 ␮lofo-phenylenediamine dihydro- chloride solution (35) were added to each well as a chromogen. Following Microscopic phagocytosis assay with Klebsiella pneumoniae a 30 min incubation (at 22¡C) in the dark, the absorbance at 450 nm was K. pneumoniae 43816, serotype 2, was obtained from the American Type evaluated with an automated reader (VERSAmax; Molecular Devices, Culture Collection (Rockville, MD) and aliquots grown in tryptic soy broth Sunnyvale, CA). The number of sRBCs per well was derived from absorbance data at 450 nm using the standard curve. The PI (number of ingested at 37¡C (5% CO2) until mid-log phase. The concentration of bacteria in culture was determined spectrophotometrically (600 nm) (32). Bacteria sRBCs) represented the number of sRBCs in an experimental well (in- were opsonized in RPMI 1640 containing 2% specific rat anti-K. pneu- gested plus adhered sRBCs) minus the mean number of sRBCs in the moniae serum as previously described (33). Rat AMs (105/well) were cytochalasin D-treated wells (adhered sRBCs) and was expressed as a per- plated and cultured overnight in glass eight-well BD Falcon tissue culture centage of the control. Independent experiments were performed in slides (BD Biosciences, Bedford, MA) as previously described. Cells were septuplet. preincubated with compounds of interest at the concentrations and times indicated in figure legends before infection. Following preincubation, se- Measurement of intracellular cAMP and PGE production rum-opsonized K. pneumoniae bacteria were added (multiple of infection 2 (MOI) ϭ 10:1) and cultures were incubated for an additional 30 min at Rat AMs were cultured overnight in six-well plates in RPMI 1640 at con- 37¡C. Because phagocytosis was directly proportional to the MOI, prelim- centrations of 3 ϫ 106 cells/well. For cAMP experiments, cultures were inary experiments were performed to determine the optimal MOI (data not then incubated for 15 min in the presence or absence of compounds of shown). Experiments were terminated and uningested bacteria removed by interest. Culture supernatants were aspirated and the cells were lysed by aspirating supernatants and washing three times with cold PBS. Slides were incubation for 20 min with 0.1 M HCl (22¡C), followed by disruption using subsequently stained with a modified Wright-Giemsa stain and examined a cell scraper. Intracellular cAMP levels were determined by enzyme- under light microscopy. Phagocytosis was quantified as previously de- linked immunosorbent assay kit according to the manufacturer (Cayman

scribed (33). The phagocytic index (PI) was determined by multiplying the Chemicals). For PGE2 determination, cells were incubated with immune- percentage of AMs containing one or more bacterium by the mean number serum opsonized K. pneumoniae (MOI ϭ 10:1) or medium alone for 60

of bacteria per positive cell. The ability to distinguish intracellular from min. Culture supernatants were collected and PGE2 levels quantiﬁed by surface-associated bacteria was veriﬁed by comparing the PI of untreated enzyme-linked immunosorbent assay according to the manufacturer (Assay AM monolayers with that of cells exposed for 45 min to the phagocytosis Designs, Ann Arbor, MI). The Journal of Immunology 561

Immunoblot analysis 3% maximal inhibition, respectively). The specificity of this effect Western blots were performed as previously described (37). Briefly, whole was demonstrated by the fact that an alternative prostanoid, the cell protein extracts were obtained by lysing freshly harvested AMs in a thromboxane A2 receptor agonist U-46619, failed to inhibit phago- ␮ buffer (50 mM Tris-HCl (pH 7.4), 25 mM KCl, 5 mM MgCl2, 0.2% Non- cytosis at concentrations up to 1 M (data not shown). As illus- idet P-40) supplemented with protease inhibitors (Roche Diagnostics, trated (Fig. 1), PGE also inhibited AM ingestion of IgG-E. coli in ␮ 2 Mannheim, Germany). Protein samples (20 g) were resolved on 10% a dose-dependent manner. Tris-HCl polyacrylamide gels and subsequently transferred to a nitrocel- lulose membrane. Membranes were probed with commercially available rabbit polyclonal EP receptor Abs (Alpha Diagnostic International, San Endogenous PGE2 restricts AM phagocytosis Antonio, TX, and Cayman Chemicals) followed by HRP-conjugated anti- Because AMs are capable of PGE2 biosynthesis, it was of interest rabbit secondary Abs and ECL Plus detection reagents (Amersham Bio- to ascertain whether levels of PGE generated endogenously in- sciences, Piscataway, NJ). 2 fluenced phagocytosis. We initially assessed the effects of a 60 min

Cell viability challenge with immune serum-opsonized K. pneumoniae on PGE2 ϭ All experimental compounds and vehicles showed no adverse effects on rat synthesis by rat AMs (MOI 10:1). Under these conditions, basal Ϯ AM viability using a commercially available MTT assay kit (Sigma-Al- production of PGE2 by AMs (183.8 4.2 pg/million AM) was drich; data not shown). only marginally enhanced following infection (205.5 Ϯ 4.5 pg/ million AM; p Ͻ 0.05, n ϭ 3). Greater enhancement of AM PGE Statistical analysis 2 production was observed with either a larger MOI (100:1) or a Data are represented as mean Ϯ SE and were analyzed with the Prism 3.0 longer incubation time for bacterial/AM cultures (data not shown). statistical program (GraphPad Software, San Diego, CA). Comparisons When endogenous PGE production was inhibited with the COX- Downloaded from between two experimental groups were performed with Student’s t test. 2 Comparisons among three or more experimental groups were performed 1/COX-2 inhibitor indomethacin, phagocytosis of K. pneumoniae with ANOVA followed by either Dunnett’s or Tukey’s multiple compar- was enhanced by 43 Ϯ 4.9% ( p Ͻ 0.05) and the ingestion of ison tests as indicated. Differences were considered significant if p Յ 0.05. IgG-opsonized sRBCs by 44 Ϯ 5.8% ( p Ͻ 0.01; Fig. 2). Because All experiments were performed on at least three separate occasions unless indomethacin has been found to have effects on cell function that otherwise specified. are independent of its COX inhibitory action (38), we examined Results the influence of two other nonspecific COX inhibitors (aspirin and http://www.jimmunol.org/ ibuprofen) on FcR-mediated phagocytosis of IgG-sRBCs (Fig. 2). PGE inhibits FcR-mediated phagocytosis 2 As illustrated, similar effects were seen with these diverse COX

Exogenous PGE2 has been shown to inhibit FcR-mediated phago- inhibitors. cytosis of IgG-opsonized sRBCs by murine AMs (27). We sought to confirm this finding with rat AMs, which were pretreated for 5 PGE2 enhances intracellular cAMP in AMs min with increasing concentrations of PGE before incubation 2 PGE2 acts through four distinct G protein-coupled receptor sub- with IgG-opsonized sRBCs. PGE2 showed a concentration-depen- types (EP1, EP2, EP3, and EP4), with distinct signaling pathways, dent suppression of FcR-mediated phagocytosis that reached but neither the PGE2 signaling mechanisms nor the EP receptor ϳ54% at 1 ␮M (Fig. 1). Because the effect of PGE on AM phago- by guest on September 25, 2021 2 expression profile of AMs is known. Because the Gs-coupled EP2 cytosis of bacterial pathogens has not been determined previously, and EP4 receptors activate, and the Gi-coupled EP3 receptor sup- we examined the ingestion of live, immune serum-opsonized K. presses adenylate cyclase activity (22), we measured changes in pneumoniae bacteria following exposure to PGE2. Phagocytosis, as measured by light microscopy, was inhibited by PGE2, with maximal inhibition (71 Ϯ 5%) found at 1 ␮M (Fig. 1). The percentage of AMs ingesting bacteria was reduced to a greater degree than was the mean number of bacteria per cell (61 Ϯ 7% vs 27 Ϯ

FIGURE 2. Effect of endogenous prostanoids on phagocytosis. Rat AMs were harvested by lung lavage and cultured as described in Materials and Methods. A, Cells were pretreated with the COX inhibitor indomethacin (10 ␮M) or vehicle for 30 min and then challenged with 2% immune

FIGURE 1. Inhibition of phagocytosis by PGE2. Rat AMs were har- serum (IS)-opsonized, live K. pneumoniae. Data represent the mean of vested by lung lavage and cultured as described in Materials and Methods. three independent experiments performed in quadruplicate. B, Cells were ␮ ␮ Cells were pretreated with PGE2 or vehicle control for 5 min and then pretreated with indomethacin (10 M), ibuprofen (100 M), aspirin (200 challenged with IgG-opsonized sRBCs (‚), 2% immune serum (IS)-opso- ␮M), or vehicle for 30 min followed by IgG-opsonized sRBCs. Data rep- nized, live K. pneumoniae (Ⅺ), or FITC-labeled, IgG-opsonized E. coli resent the mean of six independent experiments (indomethacin) and a sin- (E). PIs were calculated as described and expressed as a percentage of the gle experiment (ibuprofen and aspirin) performed in septuplet. The PI was Ͻ ءء Ͻ ء control value to which no PGE2 was added. , p 0.05, , p 0.01 com- calculated as described and expressed as a percentage of control to which -p Ͻ 0.05, by Student’s t test (A) or ANOVA fol ,ء .pared with control, by ANOVA followed by Dunnett’s multiple comparison no drug was added test (n ϭ 3Ð8 independent experiments performed in quadruplicate). lowed by Dunnett’s multiple comparison test (B). 562 PGE2 SUPPRESSION OF AM PHAGOCYTOSIS

FIGURE 3. Effect of PGE2 on intracellular cAMP concentration

([cAMP]i). A, Rat AMs were pretreated with PGE2 or vehicle control for FIGURE 5. Increasing intracellular cAMP concentration ([cAMP]i) lev- 15 min and intracellular cAMP concentration determined by enzyme im- els correlate with diminished phagocytosis. Rat AMs were preincubated ␮ munoassay as described in Materials and Methods. B, Rat AMs were pre- with either 100 nM PGE2 (A)or100 M forskolin (B) in the presence or ␮ ␮ ␮ treated with PGE2 (1 M), the EP2 receptor agonist butaprost (1 M), the absence of 10 M rolipram. Cells were then challenged with E. coli op- EP4 receptor agonist ONO-AE1-329 (1 ␮M), or vehicle control for 15 min sonized with speciﬁc rabbit polyclonal IgG to assess phagocytosis (f)or followed by determination of intracellular cAMP concentration. Data are lysed to measure intracellular cAMP concentration (Ⅺ) as described in p Ͻ 0.001 compared with vehicle Downloaded from ,ءء ,p Ͻ 0.05 ,ء .expressed as fold change from the control value, to which only vehicle was Materials and Methods p Ͻ 0.001 compared with control by ANOVA control by ANOVA followed by Dunnett’s multiple comparison test (n ϭ ,ءء ,p Ͻ 0.05 ,ء .added followed by Dunnett’s multiple comparison test (n ϭ 3Ð7 independent 3 independent experiments performed in duplicate for cAMP or quadru- experiments performed in duplicate). plicate in phagocytosis).

intracellular cAMP in response to PGE2 (Fig. 3A). Rat AMs sponse experiments demonstrated that 100 ␮M forskolin enhanced http://www.jimmunol.org/ treated for 15 min with PGE2 showed a concentration-dependent intracellular cAMP production in rat AMs to approximately the ␮ increase in intracellular cAMP, suggesting the participation of EP2 same degree as 1 M PGE2 (data not shown). A standard concen- ␮ and/or EP4 receptors. Using the highly selective EP2 and EP4 tration of rolipram was used (10 M) (39). Both PGE2 (Fig. 5A) receptor agonists butaprost and ONO-AE1-329, respectively, we and forskolin (Fig. 5B) suppressed phagocytosis. The effects of found that only the EP2 receptor agonist stimulated cAMP pro- both compounds were enhanced when cells were pretreated with duction at concentrations as high as 1 ␮M (Fig. 3B). The basal rolipram. Furthermore, inhibition of phagocytosis directly corre- intracellular concentration of cAMP in AMs (138.1 Ϯ 11.0 fmol/ lated with measured levels of cAMP. 106 cells) was not reduced by the EP1/EP3 receptor agonist sulprostone, however sulprostone (1 ␮M) limited the increase in in- An EP2 receptor agonist inhibits AM phagocytosis by guest on September 25, 2021 tracellular cAMP stimulated by butaprost (1 ␮M) by ϳ25% (data To characterize the participation of the EP2 receptor in determin- not shown). ing the effect of PGE2 on AM function, we assessed the ingestion of IgG-E. coli in the presence or absence of PGE , the EP2 agonist AMs express EP2 and EP3 receptors 2 butaprost, and the DP1/EP1/EP2 receptor antagonist AH-6809.

The expression of all four EP receptors in rat AMs was examined Butaprost and PGE2 demonstrated equivalent inhibition of phago- using commercially available polyclonal Abs. Protein preparations cytosis at a concentration of 1 ␮M (Fig. 6). The effect of butaprost from lysates of freshly harvested, unstimulated rat AMs were sub- was completely abrogated by AH-6809 (100 ␮M). The ability of jected to Western blot analysis for the detection of EP receptor protein. Neither EP1 nor EP4 receptors were detected by this meth- od; however, bands of 68 kDa and 62 kDa were seen for EP2 and EP3, respectively (Fig. 4).

Pharmacological activation of adenylate cyclase suppresses phagocytosis Our results support the ﬁndings of other investigators (28, 39) that elevations in intracellular cAMP are associated with suppression of phagocyte function. We explored the causal relationship be-

tween PGE2-induced cAMP generation and depressed phagocytosis in AM pharmacologically. We used forskolin, a direct activator of adenylate cyclase, and rolipram, an inhibitor of the phosphodiesterase IV enzyme that degrades cAMP. Preliminary dose-re-

FIGURE 6. The EP2 receptor mediates the suppressive effect of PGE2 on AM phagocytosis. Rat AMs were preincubated for 30 min with the EP2 ␮ antagonist AH-6809 (100 M) or vehicle, followed by PGE2, butaprost (EP2 agonist), sulprostone (EP3 agonist), or ONO-AE1-329 (EP4 agonist) FIGURE 4. Detection of EP receptors by Western blot analysis. Whole at a ﬁnal concentration of 1 ␮M each for 5 min. Phagocytosis was assessed cell lysates from rat AMs were prepared and Western blots performed with E. coli opsonized with speciﬁc rabbit polyclonal IgG as described in p Ͻ 0.001 vs control, ‡, p Ͻ 0.001 vs no ,ءء .using rabbit polyclonal anti-EP receptor Abs (Alpha Diagnostic Interna- Materials and Methods tional, San Antonio, TX) as described in Materials and Methods. One of AH-6809 added, by ANOVA followed by Tukey’s multiple comparison four representative Western blots is shown. test (n ϭ 3 independent experiments performed in quadruplicate). The Journal of Immunology 563

other species or from peritoneal macrophages, exhibit little or no phagocytosis of complement-opsonized targets (42). The effects of

PGE2 on AM phagocytosis via FcR-independent pathways await further investigation. Using three different in vitro models of FcR- mediated phagocytosis, we have demonstrated a dose-dependent

inhibitory effect of exogenous PGE2 on rat AM ingestion of serum- or IgG-opsonized targets (Fig. 1). Using a microscopic assay of phagocytosis we found ϳ70% inhibition of phagocytosis of live, ␮ immune serum-opsonized K. pneumoniae with 1 M PGE2.Be- cause microscopy is labor intensive and susceptible to subjective human error, we adopted and optimized a ﬂuorometric assay with greater throughput capacity and objectivity (43). Although this assay required a higher MOI (30:1) than we used for microscopy

(MOI 10:1), we still found significant inhibition with PGE2.Itis notable that the maximal degree of PGE2 inhibition observed var- FIGURE 7. Effect of PGE2 and forskolin on phagocytosis by AMs from ied among the three models used. The reasons for this variability EP2 null mice. AMs were harvested from EP2 knockout mice or wild-type are unclear but may reflect differences among the phagocytic tar- controls as described in Materials and Methods. Cells were pretreated with gets used, different target to AM ratios, the source/type of opsonin PGE (1 ␮M), forskolin (100 ␮M), or vehicle control for 5 min and then 2 used, and differences in assay sensitivity. Consistent with our ex- Downloaded from challenged with E. coli opsonized with specific rabbit polyclonal IgG. PIs perience, the maximal inhibitory effect of PGE on phagocytosis in were calculated as described and expressed as a percentage of control to 2 p Ͻ 0.001 compared with other in vitro monocyte/macrophage systems has ranged from ,ءء ,p Ͻ 0.05 ,ء .which only vehicle was added ϳ ϳ control, by ANOVA followed by Dunnett’s multiple comparison test. The 25% to 85% (23, 27Ð29). results of one experiment representative of three independent experiments Macrophages are an important source of PGE2 at sites of in- performed in quadruplicate are shown. flammation, and endogenously produced PGE2 can regulate mac-

rophage function in an autocrine fashion. In support of this, we http://www.jimmunol.org/ found that inhibition of COX activity with indomethacin, ibupro- AH-6809 to prevent the inhibitory action of butaprost was dose- fen, or aspirin revealed a suppressive effect of endogenous prosta- dependent (data not shown). Inhibition by PGE2 was not entirely noids on the phagocytic activity of AMs. It would be expected that prevented by AH-6809, but the residual degree of inhibition this role of endogenous PGE2 could be magnified in circumstances (16.9 Ϯ 4.1%) was not statistically significant as compared with characterized by overproduction of PGE2, such as cancer, aging, the control value ( p Ͼ 0.05). EP2 receptor blockade with AH- HIV infection, and malnutrition (16Ð19). 6809 did not significantly enhance the phagocytic activity of AMs. Despite the observation that PGE2 tends to suppress phagocy- Neither the EP1/EP3 agonist sulprostone (1 ␮M) nor the EP4 ag- tosis, very little is known about the receptor-mediated intracellular onist ONO-AE1-329 (1 ␮M) affected phagocytosis significantly by guest on September 25, 2021 signaling mechanisms responsible for this effect. PGE2 has been compared with control (Fig. 6). shown to stimulate AM adenylate cyclase activity (44) and agents that enhance intracellular cAMP production generally inhibit Murine AMs lacking the EP2 receptor are protected from PGE 2 phagocytosis (28, 39, 45, 46). In human peripheral blood mono- inhibition cyte-derived macrophages, for example, PGE2 suppression of A complementary approach to determining the role of the EP2 phagocytosis was associated with increased intracellular cAMP

receptor in mediating the effects of PGE2 on phagocytosis used generation (28). These cells express both the EP2 and EP4 receptor mice genetically deﬁcient in the EP2 receptor. AMs harvested as evidenced by pharmacological and genetic analysis (47, 48). from EP2 knockout mice and their wild-type strain controls were That intracellular cAMP production in AMs was stimulated in a ␮ incubated in the presence or absence of PGE2 (1 M) 5 min before dose-dependent manner by PGE2 (Fig. 3) suggested this mecha- the administration of IgG-E. coli (Fig. 7). PGE2 inhibited phago- nism for its inhibitory effect. This possibility was strongly sup- Ϯ Ͻ ϭ cytosis by wild-type AMs by 24.6 3.0% ( p 0.05; n 3) but ported by the ﬁnding that inhibition of phagocytosis by PGE2 and had no effect on EP2 knockout cells. Inhibition of AM phagocy- forskolin was augmented when cAMP degradation was prevented tosis by forskolin was equivalent in wild-type and EP2 knockout by the phosphodiesterase inhibitor rolipram. A cAMP-dependent

mice, demonstrating that the receptor null cells had intact postre- mechanism of PGE2 immunosuppression suggests the participa- ceptor signaling. tion of the EP2 and/or EP4 receptors and a possible counter-reg-

ulatory role for the Gi-coupled EP3 receptor. Using pharmacolog- Discussion ical agonists for all four EP receptors, we showed a signiﬁcant

The present study conﬁrms that PGE2 has important immunosup- increase in cAMP only with the EP2 speciﬁc agonist butaprost,

pressive effects on AM antimicrobial function (27, 40, 41) and suggesting that the EP4 receptor does not mediate PGE2 regulation enhances our understanding of the mechanisms whereby these ef- in these cells. Sulprostone, an EP1/EP3 agonist, failed to suppress

fects occur. PGE2 has previously been shown to either stimulate or basal cAMP production, although it blunted the cAMP increase inhibit the phagocytic capacity of monocytes/macrophages, with evoked by butaprost, suggesting the presence and modest func- the majority of studies demonstrating inhibition (23Ð29). In 1991, tional participation of this receptor. The role of EP2 in the sup-

Canning et al. (27) demonstrated that exogenously added PGE2 pression of AM phagocytosis was conﬁrmed using butaprost in the suppressed the phagocytosis of IgG-opsonized sRBCs by murine presence or absence of AH-6809, an effective but relatively non-

AMs. However, neither the effects of PGE2 on AM phagocytosis speciﬁc EP2 receptor antagonist. This compound, which at the

of bacterial pathogens nor the expression proﬁle of EP receptors by concentration used in this study blocked the PGE2-induced pro- AMs is known. duction of cAMP in COS cells transfected with the human EP2

Our study focused on the effects of PGE2 on phagocytosis me- receptor (49), entirely prevented inhibition of phagocytosis by ␥ diated by the FcR because rat AMs, as compared with AMs from butaprost and largely abrogated the response to PGE2. 564 PGE2 SUPPRESSION OF AM PHAGOCYTOSIS

The pharmacological evidence for PGE2-EP2 signaling was ent from the theoretical molecular masses predicted from the pri- supported by experimental data using AMs harvested from EP2 mary amino acid sequences (40 kDa and 40Ð45 kDa, respectively) knockout mice. As demonstrated in Fig. 7, EP2 knockout macro- (22, 54). This discrepancy between predicted and apparent molec- phages demonstrated phagocytic ability comparable to wild-type ular masses by SDS-PAGE has been reported previously with controls. Whereas the ingestion of IgG-E. coli by wild-type AMs membrane receptors, including rat, bovine, and human EP recep- was signiﬁcantly inhibited by PGE2, AMs from EP2 knockout tors (55Ð57), and may result from receptor oligomerization (58) or mice were not affected by exogenous PGE2. Importantly, forskolin posttranslational modiﬁcations such as glycosylation and/or phos- suppressed phagocytosis of wild-type and EP2 knockout cells phorylation (55, 56). In agreement with our results, apparent mo- equivalently, demonstrating intact cAMP signaling pathways in bilities of 68 and 62 kDa have also been reported for the human both groups of mice. EP2 and EP3 receptors (55).

Our data suggest that endogenously generated PGE2 suppresses Our findings are important given the critical role AMs play in AM phagocytosis via the EP2 receptor. However, neither pretreat- the innate immune defense against bacterial pneumonia. Impair- ment of rat AMs with AH-6809 nor deletion of the EP2 receptor in ments in the phagocytic ability of AMs increase the risk of pul- murine AMs enhanced phagocytosis in a similar manner to COX monary infection or its resultant morbidity and mortality (59). inhibitor treatment. The reasons for this are unclear. It is possible PGE2 is a prominent metabolite of AA whose production is dra- that the suppressive effect of COX inhibitors on prostanoids other matically up-regulated at sites of infection and inflammation, in- than PGE2 is responsible for the enhanced phagocytosis seen with cluding the lung (15, 60). Sources of this PGE2 include both AMs these agents. Indeed, prostacyclin-induced cAMP production in- themselves as well as neighboring cells such as epithelial cells Downloaded from hibited phagocytosis by rat peritoneal macrophages (50); however, (61). That AM function may be negatively regulated by PGE2 fits

AM capacity for prostacyclin synthesis is quite limited (51). COX- with the emerging conception of PGE2 as a suppressor of macro- inhibition might also augment phagocytosis by shunting AA away phage function (5, 6, 60). from COX enzymes and into alternative metabolic pathways such In summary, we have shown that exogenously supplied and as the 5-lipoxygenase cascade. This is important because products endogenously produced PGE2 play an inhibitory role in FcR- of the 5-lipoxygenase pathway (namely leukotriene B4 and the mediated AM phagocytosis of bacterial pathogens. This immuno- cysteinyl leukotrienes) have been found to enhance FcR-mediated suppressive action is mediated by increased cAMP levels follow- http://www.jimmunol.org/ phagocytosis in AMs (33). However, we have previously reported ing ligation of the transmembrane EP2 receptor. These results have that shunting of AA from the COX cascade into the 5-lipoxygen- important implications for future efforts to prevent and treat ase pathway did not occur when indomethacin-treated AMs were bacterial pneumonia, particularly in those subsets of immunosup- stimulated with calcium ionophore (52). When we measured PGE2 pressed individuals that may have exaggerated PGE2 production. and leukotriene B4 by AMs challenged with immune serum-opso- Variations among individuals in PGE2 production as well as EP2 nized K. pneumoniae, we similarly did not see AA shunting fol- expression might influence the susceptibility to and outcome of lowing indomethacin exposure (data not shown). In the absence of respiratory infections. Moreover, EP2 antagonism might represent measurable AA shunting, it could still be hypothesized that block- a strategy for immunostimulation. by guest on September 25, 2021 ing the endogenous suppressor compound PGE2 increases the ability of leukotrienes to promote FcR-mediated ingestion. Studies to Acknowledgments address potential interactions between PGE2 and leukotrienes and We thank Dr. Shuh Narumiya (Kyoto University, Kyoto, Japan) for avail- the relative contribution of these immunomodulators in regulating ability of the EP2 knockout mice, Dr. Takayuki Maruyama (Minase Re- AM antimicrobial function are ongoing. search Institute, ONO Pharmaceutical, Osaka, Japan) for provision of EP2 Yet another potential mechanism whereby COX inhibitors knockout mice as well as EP agonists, Dr. Bethany Moore for assistance might influence AM function includes COX-independent actions with mouse breeding, and Susan Phare and Teresa Marshall for technical of these drugs (such as effects on transcription factors and cytokine assistance. production) (38, 53). We attempted to limit this potential con- founder by 1) using chemically diverse COX inhibitors, 2) using References concentrations of these compounds below those at which nonspe- 1. Arias, E., and B. L. Smith. 2003. Deaths: preliminary data for 2001. National cific effects arise, and 3) exposing AMs to these drugs for periods Vital Statistics Report. 51:1. 2. Murray, C. J., and A. D. Lopez. 1997. Mortality by cause for eight regions of the of time short enough to minimize changes in cellular gene world: Global Burden of Disease Study. Lancet 349:1269. expression. 3. Lohmann-Matthes, M. L., C. Steinmuller, and G. Franke-Ullmann. 1994. Pul- Conversely, the failure of AH-6809 to enhance phagocytosis monary macrophages. Eur. Respir. J. 7:1678. 4. Peters-Golden, M., and M. Coffey. 1999. Role of leukotrienes in antimicrobial may result from its lack of specificity for the EP2 receptor, par- host defense of the lung. Clin. Rev. Allergy Immunol. 17:261. ticularly at the concentration used in this study (100 ␮M) (22). 5. Tilley, S. L., T. M. Coffman, and B. H. Koller. 2001. Mixed messages: modu- AMs from EP2 knockout mice tended to phagocytose IgG-E. coli lation of inflammation and immune responses by prostaglandins and thrombox- anes. J. Clin. Invest. 108:15. better than did wild-type cells, but the difference was not signifi- 6. Goodwin, J. S., and D. R. Webb. 1980. Regulation of the immune response by cant. Unexpected results with the EP2 null macrophages could prostaglandins. Clin. Immunol. Immunopathol. 15:106. occur if compensatory changes in PGE production or EP1, EP3, 7. Downey, G. P., R. S. Gumbay, D. E. Doherty, J. F. LaBrecque, J. E. Henson, 2 P. M. Henson, and G. S. Worthen. 1988. Enhancement of pulmonary inflamma- or EP4 expression are present; this remains to be investigated. tion by PGE2: evidence for a vasodilator effect. J. Appl. Physiol. 64:728. Detection of EP receptor proteins in whole cell lysates prepared 8. McCoy, J. M., J. R. Wicks, and L. P. Audoly. 2002. The role of prostaglandin E2 from rat AMs was performed using immunoblot analysis with receptors in the pathogenesis of rheumatoid arthritis. J. Clin. Invest. 110:651. 9. Armstrong, R. A. 1995. Investigation of the inhibitory effects of PGE2 and se- commercially available polyclonal Abs directed against each of the lective EP agonists on chemotaxis of human neutrophils. Br. J. Pharmacol. four EP receptors. Our results demonstrated evidence for the pres- 116:2903. 10. McLeish, K. R., G. T. Stelzer, and J. H. Wallace. 1987. Regulation of oxygen ence of the EP2 and EP3 receptors. We were unable, however, to radical release from murine peritoneal macrophages by pharmacologic doses of identify either the EP1 or EP4 receptor proteins. Our findings were PGE2. Free Radical Biol. Med. 3:15. similar using EP receptor Abs from two different commercial ven- 11. Xing, M., S. Post, R. S. Ostrom, M. Samardzija, and P. A. Insel. 1999. Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a dors (data not shown). The apparent mobilities for the EP2 and negative feedback loop for P2Y receptor activation in Madin-Darby canine kid- EP3 receptors (ϳ68 kDa and ϳ62 kDa, respectively) were differ- ney D1 cells. J. Biol. Chem. 274:10035. The Journal of Immunology 565

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